Limk2 inhibitors, compositions comprising them, and methods of their use

ABSTRACT

Inhibitors of LIM kinase 2 are disclosed, along with pharmaceutical compositions comprising them and methods of their use. Particular compounds are of the formula:

This application claims priority to U.S. provisional patent applicationNo. 61/046,602, filed Apr. 21, 2008, the entirety of which isincorporated herein by reference.

1. FIELD OF THE INVENTION

This invention relates to kinase inhibitors, compositions comprisingthem, and methods of their use to treat various diseases and disorders.

2. BACKGROUND

Protein kinases are a class of enzymes that catalyze the transfer of theγ-phosphate group from ATP to a recipient protein. The human genome isestimated to encode in excess of 500 distinct protein kinases, of whichmany have been implicated in a wide range of diseases and disorders,including cancer and inflammation.

The LIM kinases (LIMK) have been linked to the p53 pathway. See, e.g.,International Application No. WO 02/099048. LIMK belongs to a smallsubfamily of kinases with a unique combination of two N-terminal LIMmotifs and a C-terminal protein kinase domain. These LIM motifs andkinase domains are linked by a proline- and serine-rich regioncontaining several putative casein kinase and map kinase recognitionsites. LIM kinases and their pathway proteins are believed to contributeto Rho-induced reorganization of the actin cytoskeleton. Id. Members ofthe LIM kinase family include LIM kinase 1 (LIMK1) and LIM kinase 2(LIMK2). Both phosphorylate cofilin and regulates Rho family-dependentactin cytoskeletal rearrangement. Id.

LIM kinase inhibitors have been proposed for the treatment of cancer.Id. It has also been suggested that LIMK inhibitors may be useful intreating glaucoma by promoting actin depolymerization in trabecularcells and lowering ocular tension. See International Application No. WO04/047868. Current glaucoma therapies operate by different mechanisms.Prostaglandin F2a analogues (e.g., latanoprost) effect an intraocularpressure (IOP) independent increase in fluid outflow from the eye.Carbonic anhydrous inhibitors (e.g., acetazolamide), beta-blockers(e.g., timolol), sympathomimetics (e.g., pilocarpine), and alphaadrenergic receptor agonists (e.g., brimonidine) decrease aqueous humorproduction.

An enormous number of compounds, with a wide variety of chemotypes, havebeen reported as kinase inhibitors. For example, phenyl-substitutedpyrimidine compounds have been disclosed that are reportedly useful asLIMK inhibitors. See International Application WO 2006/084017.Pyrrole[2,3-d]pyrimidine-based compounds have been disclosed asinhibitors of various different kinases. See, e.g., U.S. patentpublication no. 2004/0058922; International Application WO 2007/125325;International Application WO 2007/125321; International Application WO2006/046024; U.S. patent publication no. 2005/0130954; and U.S. patentpublication no. 2006/0189638.

3. SUMMARY OF THE INVENTION

This invention is directed, in part, to compounds of the formula:

and pharmaceutically acceptable salts thereof, the substituents of whichare defined herein. Particular compounds are potent inhibitors of LIMK2.

One embodiment of the invention encompasses pharmaceutical formationscomprising compounds disclosed herein.

Another embodiment encompasses methods of using the compounds disclosedherein for the treatment, management and prevention of various diseasesand disorders affected by LIMK2, including cancer, inflammatory diseasesand disorders, and disease and disorders affecting vision (e.g.,diseases and disorders of the eye), such as glaucoma, neurodegenerationand infection.

4. BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows the effect of two compounds in the ocular hypertensionassay described in the Examples below. Compound A is a compound of theinvention, dosed at 3 μg per eye. The other compound, timolol, was dosedat 15 μg per eye. Data for the vehicle control is an average of eightexperiments using different vehicles. Data for compound A is an averageof eight experiments using different vehicles. Data for timolol is anaverage of two experiments obtained using a commercial formulation.

5. DETAILED DESCRIPTION

This invention is based, in part, on the discovery of novel compounds,which are inhibitors of LIM kinase 2 (LIMK2). LIMK2 inhibitors may beused to treat, manage and/or prevent a variety of diseases and disordersof the eye, such as glaucoma. See, e.g., U.S. patent application Ser.No. 12/188,515, filed Aug. 8, 2008. Preferred compounds of thisinvention are stable in solution, and may therefore be incorporated intoliquid dosage forms such as eye drops. In addition, particular compoundsare water soluble.

5.1. Definitions

Unless otherwise indicated, the term “alkenyl” means a straight chain,branched and/or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 10 or2 to 6) carbon atoms, and including at least one carbon-carbon doublebond. Representative alkenyl moieties include vinyl, allyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl,3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl and3-decenyl.

Unless otherwise indicated, the term “alkoxy” means an —O-alkyl group.Examples of alkoxy groups include, but are not limited to, —OCH₃,—OCH₂CH₃, —O(CH₂)₂CH₃, —O(CH₂)₃CH₃, —O(CH₂)₄CH₃, and —O(CH₂)₅CH₃.

Unless otherwise indicated, the term “alkyl” means a straight chain,branched and/or cyclic (“cycloalkyl”) hydrocarbon having from 1 to 20(e.g., 1 to 10 or 1 to 4) carbon atoms. Alkyl moieties having from 1 to4 carbons are referred to as “lower alkyl.” Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyland dodecyl. Cycloalkyl moieties may be monocyclic or multicyclic, andexamples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andadamantyl. Additional examples of alkyl moieties have linear, branchedand/or cyclic portions (e.g., 1-ethyl-4-methyl-cyclohexyl). The term“alkyl” includes saturated hydrocarbons as well as alkenyl and alkynylmoieties.

Unless otherwise indicated, the term “alkylaryl” or “alkyl-aryl” meansan alkyl moiety bound to an aryl moiety.

Unless otherwise indicated, the term “alkylheteroaryl” or“alkyl-heteroaryl” means an alkyl moiety bound to a heteroaryl moiety.

Unless otherwise indicated, the term “alkylheterocycle” or“alkyl-heterocycle” means an alkyl moiety bound to a heterocycle moiety.

Unless otherwise indicated, the term “alkynyl” means a straight chain,branched or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 20 or 2to 6) carbon atoms, and including at least one carbon-carbon triplebond. Representative alkynyl moieties include acetylenyl, propynyl,1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl,6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl,8-nonynyl, 1-decynyl, 2-decynyl and 9-decynyl.

Unless otherwise indicated, the term “aryl” means an aromatic ring or anaromatic or partially aromatic ring system composed of carbon andhydrogen atoms. An aryl moiety may comprise multiple rings bound orfused together. Examples of aryl moieties include, but are not limitedto, anthracenyl, azulenyl, biphenyl, fluorenyl, indan, indenyl,naphthyl, phenanthrenyl, phenyl, 1,2,3,4-tetrahydro-naphthalene, andtolyl.

Unless otherwise indicated, the term “arylalkyl” or “aryl-alkyl” meansan aryl moiety bound to an alkyl moiety.

Unless otherwise indicated, the terms “halogen” and “halo” encompassfluorine, chlorine, bromine, and iodine.

Unless otherwise indicated, the term “heteroalkyl” refers to an alkylmoiety (e.g., linear, branched or cyclic) in which at least one of itscarbon atoms has been replaced with a heteroatom (e.g., N, O or S).

Unless otherwise indicated, the term “heteroalkylaryl” or“heteroalkyl-aryl” refers to a heteroalkyl moiety bound to an alkylmoiety.

Unless otherwise indicated, the term “heteroalkylheterocycle” or“heteroalkylheterocycle” refers to a heteroalkyl moiety bound toheterocycle moiety.

Unless otherwise indicated, the term “heteroaryl” means an aryl moietywherein at least one of its carbon atoms has been replaced with aheteroatom (e.g., N, O or S). Examples include, but are not limited to,acridinyl, benzimidazolyl, benzofuranyl, benzoisothiazolyl,benzoisoxazolyl, benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl,imidazolyl, indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl,phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl, tetrazolyl, thiazolyl,and triazinyl.

Unless otherwise indicated, the term “heteroarylalkyl” or“heteroaryl-alkyl” means a heteroaryl moiety bound to an alkyl moiety.

Unless otherwise indicated, the term “heterocycle” refers to anaromatic, partially aromatic or non-aromatic monocyclic or polycyclicring or ring system comprised of carbon, hydrogen and at least oneheteroatom (e.g., N, O or S). A heterocycle may comprise multiple (i.e.,two or more) rings fused or bound together. Heterocycles includeheteroaryls. Examples include, but are not limited to,benzo[1,3]dioxolyl, 2,3-dihydro-benzo[1,4]dioxinyl, cinnolinyl, furanyl,hydantoinyl, morpholinyl, oxetanyl, oxiranyl, piperazinyl, piperidinyl,pyrrolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl and valerolactamyl.

Unless otherwise indicated, the term “heterocyclealkyl” or“heterocycle-alkyl” refers to a heterocycle moiety bound to an alkylmoiety.

Unless otherwise indicated, the term “heterocycloalkyl” refers to anon-aromatic heterocycle.

Unless otherwise indicated, the term “heterocycloalkylalkyl” or“heterocycloalkyl-alkyl” refers to a heterocycloalkyl moiety bound to analkyl moiety.

Unless otherwise indicated, the term “LIMK2 IC₅₀” is the IC₅₀ of acompound determined using the in vitro human LIM kinase 2 inhibitionassay described in the Examples, below.

Unless otherwise indicated, the terms “manage,” “managing” and“management” encompass preventing the recurrence of the specifieddisease or disorder in a patient who has already suffered from thedisease or disorder, and/or lengthening the time that a patient who hassuffered from the disease or disorder remains in remission. The termsencompass modulating the threshold, development and/or duration of thedisease or disorder, or changing the way that a patient responds to thedisease or disorder.

Unless otherwise indicated, the term “pharmaceutically acceptable salts”refers to salts prepared from pharmaceutically acceptable non-toxicacids or bases including inorganic acids and bases and organic acids andbases. Suitable pharmaceutically acceptable base addition salts include,but are not limited to, metallic salts made from aluminum, calcium,lithium, magnesium, potassium, sodium and zinc or organic salts madefrom lysine, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. Suitable non-toxic acids include, but are not limited to,inorganic and organic acids such as acetic, alginic, anthranilic,benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic,glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phenylacetic, phosphoric, propionic, salicylic, stearic, succinic,sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid.Specific non-toxic acids include hydrochloric, hydrobromic, phosphoric,sulfuric, and methanesulfonic acids. Examples of specific salts thusinclude hydrochloride and mesylate salts. Others are well-known in theart. See, e.g., Remington's Pharmaceutical Sciences, 18^(th) ed. (MackPublishing, Easton Pa.: 1990) and Remington: The Science and Practice ofPharmacy, 19^(th) ed. (Mack Publishing, Easton Pa.: 1995).

Unless otherwise indicated, a “potent LIMK2 inhibitor” is a compoundthat has a LIMK2 IC₅₀ of less than about 250 nM.

Unless otherwise indicated, the terms “prevent,” “preventing” and“prevention” contemplate an action that occurs before a patient beginsto suffer from the specified disease or disorder, which inhibits orreduces the severity of the disease or disorder. In other words, theterms encompass prophylaxis.

Unless otherwise indicated, a “prophylactically effective amount” of acompound is an amount sufficient to prevent a disease or condition, orone or more symptoms associated with the disease or condition, orprevent its recurrence. A “prophylactically effective amount” of acompound means an amount of therapeutic agent, alone or in combinationwith other agents, which provides a prophylactic benefit in theprevention of the disease. The term “prophylactically effective amount”can encompass an amount that improves overall prophylaxis or enhancesthe prophylactic efficacy of another prophylactic agent.

Unless otherwise indicated, the term “stereoisomeric mixture”encompasses racemic mixtures as well as stereomerically enrichedmixtures (e.g., R/S=30/70, 35/65, 40/60, 45/55, 55/45, 60/40, 65/35 and70/30).

Unless otherwise indicated, the term “stereomerically pure” means acomposition that comprises one stereoisomer of a compound and issubstantially free of other stereoisomers of that compound. For example,a stereomerically pure composition of a compound having one stereocenterwill be substantially free of the opposite stereoisomer of the compound.A stereomerically pure composition of a compound having twostereocenters will be substantially free of other diastereomers of thecompound. A typical stereomerically pure compound comprises greater thanabout 80% by weight of one stereoisomer of the compound and less thanabout 20% by weight of other stereoisomers of the compound, greater thanabout 90% by weight of one stereoisomer of the compound and less thanabout 10% by weight of the other stereoisomers of the compound, greaterthan about 95% by weight of one stereoisomer of the compound and lessthan about 5% by weight of the other stereoisomers of the compound,greater than about 97% by weight of one stereoisomer of the compound andless than about 3% by weight of the other stereoisomers of the compound,or greater than about 99% by weight of one stereoisomer of the compoundand less than about 1% by weight of the other stereoisomers of thecompound.

Unless otherwise indicated, the term “substituted,” when used todescribe a chemical structure or moiety, refers to a derivative of thatstructure or moiety wherein one or more of its hydrogen atoms issubstituted with a chemical moiety or functional group such as, but notlimited to, alcohol, aldehyde, alkoxy, alkanoyloxy, alkoxycarbonyl,alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl,alkylcarbonyloxy (—OC(O)alkyl), amide (e.g. —C(O)NH-alkyl-,-alkylNHC(O)alkyl), amidinyl (e.g., —C(NH)NH-alkyl-, —C(NR)NH₂), amine(primary, secondary and tertiary such as alkylamino, arylamino,arylalkylamino), aroyl, aryl, aryloxy, azo, carbamoyl (e.g.,—NHC(O)O-alkyl-, —OC(O)NH-alkyl), carbamyl (e.g., CONH₂, CONH-alkyl,CONH-aryl, CONH-arylalkyl), carbonyl, carboxyl, carboxylic acid,carboxylic acid anhydride, carboxylic acid chloride, cyano, ester,epoxide, ether (e.g., methoxy, ethoxy), guanidino, halo, haloalkyl(e.g., —CCl₃, —CF₃, —C(CF₃)₃), heteroalkyl, hemiacetal, imine (primaryand secondary), isocyanate, isothiocyanate, ketone, nitrile, nitro, oxo,phosphodiester, sulfide, sulfonamido (e.g., SO₂NH₂), sulfone, sulfonyl(including alkylsulfonyl, arylsulfonyl and arylalkylsulfonyl),sulfoxide, thiol (e.g., sulfhydryl, thioether) and urea (e.g.,—NHCONH-alkyl-).

Unless otherwise indicated, a “therapeutically effective amount” of acompound is an amount sufficient to provide a therapeutic benefit in thetreatment or management of a disease or condition, or to delay orminimize one or more symptoms associated with the disease or condition.A “therapeutically effective amount” of a compound means an amount oftherapeutic agent, alone or in combination with other therapies, whichprovides a therapeutic benefit in the treatment or management of thedisease or condition. The term “therapeutically effective amount” canencompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of a disease or condition, or enhances thetherapeutic efficacy of another therapeutic agent.

Unless otherwise indicated, the terms “treat,” “treating” and“treatment” contemplate an action that occurs while a patient issuffering from the specified disease or disorder, which reduces theseverity of the disease or disorder, or retards or slows the progressionof the disease or disorder.

Unless otherwise indicated, the term “include” has the same meaning as“include, but are not limited to,” and the term “includes” has the samemeaning as “includes, but is not limited to.” Similarly, the term “suchas” has the same meaning as the term “such as, but not limited to.”

Unless otherwise indicated, one or more adjectives immediately precedinga series of nouns is to be construed as applying to each of the nouns.For example, the phrase “optionally substituted alky, aryl, orheteroaryl” has the same meaning as “optionally substituted alky,optionally substituted aryl, or optionally substituted heteroaryl.”

It should be noted that a chemical moiety that forms part of a largercompound may be described herein using a name commonly accorded it whenit exists as a single molecule or a name commonly accorded its radical.For example, the terms “pyridine” and “pyridyl” are accorded the samemeaning when used to describe a moiety attached to other chemicalmoieties. Thus, the two phrases “XOH, wherein X is pyridyl” and “XOH,wherein X is pyridine” are accorded the same meaning, and encompass thecompounds pyridin-2-ol, pyridin-3-ol and pyridin-4-ol.

It should also be noted that if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold ordashed lines, the structure or the portion of the structure is to beinterpreted as encompassing all stereoisomers of it. Moreover, any atomshown in a drawing with unsatisfied valences is assumed to be attachedto enough hydrogen atoms to satisfy the valences. In addition, chemicalbonds depicted with one solid line parallel to one dashed line encompassboth single and double (e.g., aromatic) bonds, if valences permit.

5.2. Compounds

This invention encompasses compounds of the formula:

and pharmaceutically acceptable salt thereof, wherein: A is optionallysubstituted aryl or heterocycle; R₁ is hydrogen, halogen, cyano, oroptionally substituted alkyl, heteroalkyl, aryl, or heterocycle; R₂ ishydrogen, halogen, cyano, or optionally substituted alkyl, heteroalkyl,aryl, or heterocycle; R₃ is R_(3A), OR_(3B), N(R_(3B))₂, NC(O)R_(3B),NC(O)OR_(3B), NC(O)N(R_(3B))₂, SR_(3B), SOR_(3B), or SO₂R_(3B); eachR_(3A) is cyano, C(O)R_(3C), CO₂R_(3C), CON(R_(3C))₂, or optionallysubstituted alkyl, alkylaryl, alkylheterocycle, aryl, heteroalkyl,heteroalkylaryl, heteroalkylheterocycle, or heterocycle; each R_(3B) isindependently hydrogen, C(O)R_(3C), CO₂R_(3C), CON(R_(3C))₂, oroptionally substituted alkyl, heteroalkyl, aryl, or heterocycle; eachR_(3C) is independently optionally substituted alkyl, aryl orheterocycle; each R₄ is independently hydrogen, halogen, OR_(4B),OC(O)R_(4B), OC(O)N(R_(4B))₂, N(R_(4B))₂, NC(O)R_(4A), NC(O)OR_(4A),NC(O)N(R_(4A)), SR_(4B), SOR_(4B), or SO₂R_(4B), or optionallysubstituted alkyl, heteroalkyl, aryl, or heterocycle; each R_(4B) isindependently hydrogen or optionally substituted alkyl, heteroalkyl,aryl, or heterocycle; and n is 0-3.

In a particular embodiment, the compound is such that: 1) each R_(4A) isindependently hydrogen or optionally substituted alkyl or heteroalkyl;and 2) when R₁ and R₂ are both hydrogen and A is phenyl, R₄ is nothalogen.

In one embodiment, A is optionally substituted phenyl. In another, A isoptionally substituted heterocycle.

In one embodiment, R₁ is hydrogen, halogen, cyano, or optionallysubstituted lower alkyl. In a particular embodiment, R₁ is hydrogen. Inanother, R₁ is halogen (e.g., chloro). In another, R₁ is optionallysubstituted lower alkyl (e.g., methyl).

In one embodiment, R₂ is hydrogen, halogen, cyano, or optionallysubstituted lower alkyl. In a particular embodiment, R₂ is hydrogen. Inanother, R₂ is halogen (e.g., chloro). In another, R₂ is optionallysubstituted lower alkyl (e.g., methyl).

In one embodiment, R₃ is R_(3A), OR_(3B), or N(R_(3B))₂. In a particularembodiment, R₃ is R_(3A) and R_(3A) is optionally substituted alkyl,alkylheterocycle, heteroalkyl, or heterocycle. In a specific embodiment,R_(3A) is optionally substituted alkyl. In another, R_(3A) is optionallysubstituted alkylheterocycle. In another, R_(3A) is optionallysubstituted heteroalkyl. In another, R_(3A) is optionally substitutedheterocycle. In a particular embodiment, R₃ is OR_(3B) or N(R_(3B))₂,and R_(3B) is hydrogen or optionally substituted alkyl or aryl. In aspecific embodiment, R_(3B) is hydrogen. In another, R_(3B) isoptionally substituted alkyl. In another, R_(3B) is optionallysubstituted aryl.

In one embodiment, R₄ is halogen. In another, R₄ is OC(O)N(R_(4A))₂ andat least one R_(4A) is, for example, lower alkyl (e.g., methyl).

Particular compounds of the invention are potent LIMK2 inhibitors.Certain compounds have a LIMK2 IC₅₀ of less than about 100, 75, 50, 25or 10 nM.

5.3. Methods of Synthesis

Compounds of the invention may be prepared by methods known in the art.See, e.g., U.S. patent publication nos. 2004/0058922 and 2005/0130954.

Pyrrolopyrimidines may be prepared by a variety of methods known in theart. See, e.g., West, J. Org. Chem. 26:4959 (1961); Aono et al., EP0733633-B1. One approach is described in U.S. patent application Ser.No. 11/876,051, filed Oct. 22, 2007, and shown below in Scheme 1:

The resulting 4-hydroxy pyrrolo[2,3-d]pyrimidine compound is thenconverted to the corresponding 4-chloro compound (compound f in Scheme2, below) using methods known in the art. See, e.g., West, J. Org. Chem.26:4959 (1961). That compound is then used to prepare compounds of theinvention, as shown below in Scheme 2:

In the method represented in Scheme 2, commercially available1-tert-butyl 4-ethyl piperidine-1,4-dicarboxylate (a) is alkylated witha suitable base (e.g., lithium diisopropyl amide) and an alklylatingagent, (e.g., an alkyl bromide or chloride), to give compound b. Othercompounds of formula b are commercially available. Saponification ofcompound b with a base such as LiOH or NaOH in aqueous solution givescarboxylic acid c, which can be coupled with amines of formula NH₂Ausing suitable reagents (e.g., HATU with diisopropylethylamine inisopropyl acetate) to give amide d. Treatment of compound d with asuitable acid (e.g., HCl in MeOH or TFA in CH₂Cl₂) removes the Boc groupto give amine e. Heating amine e and chloride f with a suitable base(e.g., diisopropylethylamine) in a suitable solvent (e.g., isopropanol,isobutanol) affords compound h. If desired, known methods can be used totransform that compound into various others encompassed by thisinvention.

5.4. Methods of Use

This invention encompasses a method of inhibiting LIMK2, which comprisescontacting LIMK2 with a potent LIMK2 inhibitor. Preferred potent LIMK2inhibitors are compounds of the invention (i.e., compounds disclosedherein).

A particular embodiment encompasses a method of treating, managing orpreventing an inflammatory disease or disorder in a patient, whichcomprises administering to the patient in need thereof a therapeuticallyor prophylactically effective amount of a compound of the invention.

Another embodiment encompasses a method of treating, managing orpreventing cancer in a patient, which comprises administering to thepatient in need thereof a therapeutically or prophylactically effectiveamount of a compound of the invention.

Another embodiment encompasses a method of lowering intraocular pressurein a patient, which comprises inhibiting LIMK2 activity or expression ina patient in need thereof. In one method, LIMK2 activity is inhibited bycontacting the eye of the patient with a potent LIMK2 inhibitor.Particular potent LIMK2 inhibitors are disclosed herein. In anothermethod, LIMK2 expression is inhibited by administering to the eye of thepatient a compound (e.g., a siRNA) that inhibits the expression ofLIMK2.

Another embodiment encompasses a method of treating, managing orpreventing a disease or disorder affecting vision in a patient, whichcomprises inhibiting LIMK2 activity or expression in a patient in needthereof. In one method, LIMK2 activity is inhibited by contacting theeye of the patient with a potent LIMK2 inhibitor. Particular potentLIMK2 inhibitors are disclosed herein. Diseases and disorders affectingvision include glaucoma, neurodegenerative diseases, and infectiousdiseases.

5.5. Pharmaceutical Formulations

This invention encompasses pharmaceutical compositions comprising one ormore compounds of the invention. Certain pharmaceutical compositions aresingle unit dosage forms suitable for oral, mucosal (e.g., nasal,sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous,intravenous, bolus injection, intramuscular, or intraarterial),transdermal, topical and ophthalmic (e.g., topical, intravitreal)administration to a patient.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a patient,including suspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The formulation should suit the mode of administration. For example,oral administration requires enteric coatings to protect the compoundsof this invention from degradation within the gastrointestinal tract.Similarly, a formulation may contain ingredients that facilitatedelivery of the active ingredient(s) to the site of action. For example,compounds may be administered in liposomal formulations, in order toprotect them from degradative enzymes, facilitate transport incirculatory system, and effect delivery across cell membranes tointracellular sites.

The composition, shape, and type of a dosage form will vary depending onits use. For example, a dosage form used in the acute treatment of adisease may contain larger amounts of one or more of the activeingredients it comprises than a dosage form used in the chronictreatment of the same disease. Similarly, a parenteral dosage form maycontain smaller amounts of one or more of the active ingredients itcomprises than an oral dosage form used to treat the same disease. Theseand other ways in which specific dosage forms encompassed by thisinvention will vary from one another will be readily apparent to thoseskilled in the art. See, e.g., Remington's Pharmaceutical Sciences,18^(th) ed. (Mack Publishing, Easton Pa.: 1990).

5.5.1. Oral Dosage Forms

Pharmaceutical compositions of the invention suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. See, e.g.,Remington's Pharmaceutical Sciences, 18^(th) ed. (Mack Publishing,Easton Pa.: 1990).

Typical oral dosage forms are prepared by combining the activeingredient(s) in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms. If desired, tablets can becoated by standard aqueous or nonaqueous techniques. Such dosage formscan be prepared by conventional methods of pharmacy. In general,pharmaceutical compositions and dosage forms are prepared by uniformlyand intimately admixing the active ingredients with liquid carriers,finely divided solid carriers, or both, and then shaping the productinto the desired presentation if necessary. Disintegrants may beincorporated in solid dosage forms to facility rapid dissolution.Lubricants may also be incorporated to facilitate the manufacture ofdosage forms (e.g., tablets).

5.5.2. Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are specifically sterileor capable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

5.5.3. Transdermal, Topical and Mucosal Dosage Forms

Transdermal, topical, and mucosal dosage forms include, but are notlimited to, ophthalmic solutions, sprays, aerosols, creams, lotions,ointments, gels, solutions, emulsions, suspensions, or other forms knownto one of skill in the art. See, e.g., Remington's PharmaceuticalSciences, 18^(th) ed. (Mack Publishing, Easton Pa.: 1990); andIntroduction to Pharmaceutical Dosage Forms, 4^(th) ed. (Lea & Febiger,Philadelphia: 1985). Transdermal dosage forms include “reservoir type”or “matrix type” patches, which can be applied to the skin and worn fora specific period of time to permit the penetration of a desired amountof active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms are well known to those skilled in the pharmaceutical arts, anddepend on the particular tissue to which a given pharmaceuticalcomposition or dosage form will be applied.

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients of the invention. For example, penetrationenhancers may be used to assist in delivering active ingredients to thetissue.

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates may also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

5.5.4. Ophthalmic Dosage Forms

Compounds of the invention can be delivered to the eye (e.g., topically)using aqueous solutions, aqueous suspensions, and ointments. As thoseskilled in the art are aware, the ophthalmic product must be sterile inits final container to prevent microbial contamination of the eye.Preservatives may be used to maintain sterility once the container hasbeen opened. Ophthalmic formulations also require that the pH, buffercapacity, viscosity, and tonicity of the formulation be controlled.Preferred formulations have a pH of from about 6.5 to 8.5, and a buffercapacity of from about 0.01 to 0.1. Particular formations are isotonic.Particular formations have a viscosity of from about 25 to 50 cps.

Ingredients that may be used to provide safe vehicles that effectivelydeliver an active pharmaceutical ingredient (API) to its site of actionare well known, but will vary depending on the physical and chemicalcharacteristics of the API.

Appropriately buffered aqueous solutions may be used for the delivery ofwater soluble compounds. In solution compositions, polymeric ingredientsare typically used to increase the composition's viscosity. Examples ofsuitable polymers include cellulosic polymers (e.g., hydroxypropylmethylcellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose),synthetic polymers (e.g., carboxyvinyl polymers, polyvinyl alcohol),polysaccharides (e.g., xanthan gum, guar gum, and dextran), and mixturesthereof. See, e.g., U.S. Pat. Nos. 4,136,173 and 7,244,440. Suspensionsmay also be used to deliver compounds. Polymeric ingredients aretypically used in suspension compositions as physical stability aids,helping to keep the insoluble ingredients suspended or easilyredispersible. Id.

Preservatives may be used to ensure the sterility of formations.Suitable preservatives include benzalkonium chloride, benzethoniumchloride, chlorobutanol, phenylmercuric acetate, phenylmercuric nitrate,thimerosal, methylparaben, and propyl-parabens. And antioxidants may beused to ensure the stability of formations susceptible to oxidation.Suitable antioxidants include ethylenediaminetetraacetic acid, sodiumbisulfite, sodium metabisulfite, and thiourea.

6. EXAMPLES

Aspects of this invention can be understood from the following examples,which do not limit its scope.

6.1. Example 1 Synthesis of1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-(3-(4-fluorophenoxy)phenyl)-4-methylpiperidine-4-carboxamide

A. Preparation of tert-butyl4-(3-(4-fluorophenoxy)phenylcarbamoyl)-4-methylpiperidine-1-carboxylate.1-(tert-butoxycarbonyl)-4-methylpiperidine-4-carboxylic acid (243 mg,1.0 mmol), 3-(4-fluorophenoxy)aniline (203 mg, 1.0 mmol), DCC (206 mg,1.0 mmol), and HOAt (138 mg, 1.01 mmol) were combined in EtOAc (50 mL)and DMF (5 mL). The reaction mixture was heated at 80° C. for 20 hours.Most of the EtOAc was evaporated, resulting in precipitation of a solid,which was removed by filtration. The filtrate was diluted with EtOAc,washed with 1 N aq. HCl, water, and brine, dried, and concentrated undervacuum to give the title compound. MS (ES+) [M+H]⁺=429.

B. Preparation ofN-(3-(4-fluorophenoxy)phenyl)-4-methylpiperidine-4-carboxamide.tert-Butyl4-(3-(4-fluorophenoxy)phenylcarbamoyl)-4-methylpiperidine-1-carboxylatefrom step A was treated with 4 N HCl in dioxane for 16 hours at roomtemperature. The product was isolated by filtration and washed withether to afford the title compound (206 mg, 56% for two steps) as thehydrochloride salt. MS (ES+) [M+H]⁺=329.

C. Preparation of 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine. To asolution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (154 mg, 1.0 mmol) inCH₂Cl₂ (6 mL) was added NCS (134 mg, 1.0 mmol). The reaction mixture wasrefluxed for 18 hours, filtered, and concentrated under vacuum to givethe title compound, which was carried on crude. MS (ES+) [M+H]⁺=189.

D. Preparation of1-(5-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-(3-(4-fluorophenoxy)phenyl)-4-methylpiperidine-4-carboxamide.N-(3-(4-Fluorophenoxy)phenyl)-4-methylpiperidine-4-carboxamide from stepB (50 mg, 0.13 mmol), 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine from stepC (19 mg, 0.1 mmol), and triethylamine (0.04 mL, 0.3 mmol) were combinedin isopropanol (1 mL) and heated at 130° C. for 30 minutes undermicrowave conditions. The product was isolated by prep HPLC to give thetitle compound.

¹H NMR (CD₃OD): δ 8.22 (s, 1H), 7.30-7.35 (m, 4H), 7.03-7.14 (m, 4H),6.73-6.76 (m, 1H), 3.96-4.02 (m, 2H), 3.44-3.51 (m, 2H), 2.35-2.38 (m,2H), 1.74-1.81 (m, 2H), 1.38 (s, 3H); MS (ES+) [M+H]⁺=480.

6.2. Example 2 Synthesis of 1-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-(3-(4-fluorophenoxy)phenyl)-4-methylpiperidine-4-carboxamide

A. Preparation of 5-bromo-4-chloro-7H-pyrrolo[23-d]pyrimidine. To asolution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (1.2 g, 7.8 mmol) inCH₂Cl₂ (25 mL) was added N-bromoacetamide (1.2 g, 8.7 mmol) in CH₂Cl₂(25 mL). The reaction mixture was refluxed for 40 minutes, thenconcentrated and washed with cold water. The crude material wasrecrystallized from a minimal amount of isopropanol and dried undervacuum to give the title compound (82%) as light-gray solid. MS (ES+)[M+H]⁺=232.

B. Preparation of1-(5-bromo-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-(3-(4-fluorophenoxy)phenyl)-4-methylpiperidine-4-carboxamide.To a solution ofN-(3-(4-fluorophenoxy)phenyl)-4-methylpiperidine-4-carboxamide fromexample 1, step B, (36 mg, 0.1 mmol) in isopropanol (1 mL), was added to5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine from step A (24 mg, 0.1mmol) and triethylamine (0.04 mL, 0.3 mmol). The reaction mixture washeated at 100° C. for 10 minutes under microwave condition, and thenconcentrated under vacuum. The product was isolated by prep HPLC to givethe title compound.

¹H NMR (CD₃OD): δ 8.21 (s, 1H), 7.22-7.35 (m, 4H), 6.98-7.17 (m, 4H),6.72-6.77 (m, 1H), 3.84-3.95 (m, 2H), 3.32-3.51 (m, 2H), 2.30-2.40 (m,2H), 1.72-1.85 (m, 2H), 1.38 (s, 3H); MS (ES+) [M+H]⁺=525.

6.3. Example 3 Synthesis ofN-(3-(4-fluorophenoxy)phenyl)-4-methyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide

To a solution ofN-(3-(4-fluorophenoxy)phenyl)-4-methylpiperidine-4-carboxamide fromexample 1, step B, (20 mg, 0.05 mmol) in isopropanol (1 mL), was added4-chloro-7H-pyrrolo[2,3-d]pyrimidine (15 mg, 0.1 mmol) and triethylamine(0.04 mL, 0.3 mmol). The reaction mixture was heated at 180° C. for 30minutes under microwave conditions, and then concentrated under vacuum.The product was isolated by prep HPLC to give the title compound.

¹H NMR (CD₃OD): δ 8.01 (s, 1H), 7.18-7.23 (m, 3H), 6.91-7.16 (m, 5H),6.65-6.68 (d, 1H), 6.51-6.53 (d, 1H), 4.12-4.25 (m, 2H), 3.351-3.62 (m,2H), 2.13-2.28 (m, 2H), 1.50-1.60 (m, 2H), 1.25 (s, 3H); MS (ES+)[M+H]⁺=447.

6.4. Example 4 Synthesis ofN-(3-tert-butylphenyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-phenylpiperidine-4-carboxamide

A. Preparation of 4-phenylpiperidine-4-carboxylic acid.1-(tert-Butoxycarbonyl)-4-phenylpiperidine-4-carboxylic acid (1.0 g, 3.3mmol) was treated with 4 N HCl in dioxane (20 mL) for 2 hours at roomtemperature. The product was isolated by filtration to provide the titlecompound (0.74 g, 93%) as a white solid. MS (ES+) [M+H]⁺=206.

B. Preparation of1-(5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-phenylpiperidine-4-carboxylicacid. To a solution of 4-phenylpiperidine-4-carboxylic acid from step A(0.74 g, 3.1 mmol) in isopropanol (10 mL), was added4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (0.52 g, 3.1 mmol) andtriethylamine (1.0 mL, 9.8 mmol). The reaction mixture was heated at180° C. for 180 minutes under microwave condition, and then concentratedunder vacuum. The residue was dissolved in ice water and neutralized topH 4 by addition of 1 N aq. HCl. The product was isolated by filtrationand dried under vacuum for 3 hours at 65° C. to give the title compound(62%) as a brown solid. MS (ES+) [M+H]⁺=336.

C. Preparation ofN-(3-tert-butylphenyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-phenylpiperidine-4-carboxamide.To a solution of1-(5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-phenylpiperidine-4-carboxylicacid from step B (33 mg, 0.1 mmol) in DMF (2 mL) was added3-tert-butylaniline (15 mg, 1.0 mmol), BOP (50 mg, 0.11 mmol) andtriethylamine (0.04 mL, 0.3 mmol). The reaction mixture was stirred atroom temperature for 20 hours, and the amide product was isolated byprep HPLC to give the title compound.

¹H NMR (CD₃OD): δ 8.20 (s, 1H), 7.16-7.57 (m, 9H), 7.01 (s, 1H),3.90-3.94 (d, 2H), 3.45-3.52 (t, 2H), 2.73-2.78 (d, 2H), 2.46 (s, 3H),2.28-2.35 (t, 2H), 1.30 (s, 9H); MS (ES+) [M+H]⁺=468.

6.5. Example 5 Synthesis of4-allyl-N-(3-chlorophenyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide

A. Preparation of tert-butyl4-allyl-4-(3-chlorophenylcarbamoyl)piperidine-1-carboxylate. Under anatmosphere of N₂,4-allyl-1-(tert-butoxycarbonyl)piperidine-4-carboxylicacid (0.158 g, 0.586 mmol), pyridine (0.189 mL, 2.34 mmol), and DMF (3drops) were dissolved in CH₂Cl₂ (10 mL) and cooled in an ice bath. Tothe cooled solution, oxalyl chloride (0.056 mL, 0.645 mmol) was addeddropwise. The reaction was stirred for 40 min, after which time3-chloroaniline (0.061 mL, 0.586 mmol) was added. The reaction wasremoved from the ice bath and stirred at room temperature overnight. Thecrude reaction was washed with 1 N aq. HCl and brine, dried over MgSO₄,and concentrated under vacuum. The residue was purified by silica gelchromatography (ethyl acetate/hexanes gradient) to give the titlecompound in 51% yield. MS (ES+) [M+H]⁺=379.

B. Preparation of 4-allyl-N-(3-chlorophenyl)piperidine-4-carboxamide. Toa solution of tert-butyl4-allyl-4-(3-chlorophenylcarbamoyl)piperidine-1-carboxylate from step A(0.172 g, 0.453 mmol) in MeOH (10 mL) was added 4 MHCl in dioxane (0.340mL, 1.36 mmol). The reaction was heated at 50° C. for 30 min, and thenconcentrated under vacuum, re-dissolved in MeOH and re-concentratedtwice to give the title compound as the hydrochloride salt. MS (ES+)[M+H]⁺=279.

C. Preparation of4-allyl-N-(3-chlorophenyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide.4-Allyl-N-(3-chlorophenyl)piperidine-4-carboxamide from step B (0.142 g,0.453 mmol), 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (0.075 g,0.453 mmol), N,N-diisopropylethylamine (0.236 mL, 1.35 mmol), andisopropanol (5 mL) were combined and heated at 120° C. overnight in apressure vessel. The crude reaction was dissolved in CH₂Cl₂, washed withH₂O and brine, dried over MgSO₄, and concentrated under vacuum. Theresidue was purified by silica gel chromatography (MeOH:CH₂Cl₂ gradient)to give the title compound in 61% yield.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.19 (s, 1H), 7.73 (s, 1H), 7.48 (d, J=8Hz, 1H), 7.32 (t, J=8, 16 Hz, 1H), 7.14 (d, J=8 Hz, 1H), 7.00 (s, 1H),5.78-5.88 (m, 1H), 5.11-5.16 (m, 2H), 3.91 (d, J=13.6 Hz, 2H), 3.24-3.30(m, 2H), 2.50 (d, J=7.6 Hz, 2H), 2.45 (s, 3H), 2.41 (d, J=13.6 Hz, 2H),1.80-1.86 (m, 2H); MS (ES+) [M+H]⁺=410.

6.6. Example 6 Synthesis ofN-(3-methoxyphenyl)-4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide

A. Preparation of 4-methylpiperidine 4-carboxylic acid.1-(tert-Butoxycarbonyl)-4-methylpiperidine-4-carboxylic acid (0.500 g,2.06 mmol) was treated with 4 M HCl in dioxane (10 mL) for 2 hours atroom temperature to afford the title compound (0.294 g, 100%) as thehydrochloride salt. MS (ES+) [M+H]⁺=144.0.

B. Preparation of4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4yl)-piperidine-4-carboxylicacid. To a solution of 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine(0.274 g, 1.64 mmol) in isopropanol (10 mL) were added4-methylpiperidine 4-carboxylic acid hydrochloric acid from step A(0.294 g, 1.64 mmol) and triethylamine (0.654 mL, 4.92 mmol). Thereaction was heated to 180° C. for 1 hour under microwave conditions,concentrated, taken up with ice water, and neutralized to pH 3 with 6 Naq. HCl to precipitate the product. The product was collected byfiltration and dried under vacuum overnight to give the title compound(0.42 g, 75%) as a gray solid. MS (ES+) [M+H]⁺=275.2.

C. Preparation ofN-(3-methoxyphenyl)-4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide.To a solution of4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4yl)-piperidine-4-carboxylicacid from step B (0.020 g, 0.073 mmol) in DMF (1 mL) were added BOPreagent (0.065 g, 0.15 mmol), triethylamine (0.019 mL, 0.15 mmol), and3-methoxyaniline (0.012 mL, 0.11 mmol). The reaction was stirredovernight, and then concentrated under vacuum. The residue was purifiedby prep HPLC to give the title compound (0.091 g, 33%).

¹H NMR (CD₃OD): δ 8.07 (1H, bs), 7.00-7.17 (3H, m), 6.89 (1H, s),6.58-6.61 (1H, m), 3.62-3.72 (5H, m), 3.22-3.34 (2H, m), 2.12-2.33 (5H,m), 1.62-1.71 (2H, m), 1.29 (3H, s); MS (ES+) [M+H]⁺=380.1.

6.7. Example 7 Synthesis of4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-p-tolylpiperidine-4-carboxamide

To a solution of4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4yl)-piperidine-4-carboxylicacid from example 6, step B, (0.020 g, 0.073 mmol) in DMF (1 mL) wereadded BOP reagent (0.065 g, 0.15 mmol), triethylamine (0.019 mL, 0.15mmol), and 4-methylaniline (0.012 mL, 0.11 mmol). The reaction wasstirred overnight and concentrated under vacuum. The residue waspurified by prep HPLC to give the title compound (0.0133 g, 50%).

¹H NMR (CD₃OD): δ 8.07 (1H, bs), 7.31 (2H, J=8.4 Hz, d), 7.04 (2H, J=8.4Hz, d), 6.89 (1H, s), 3.68-3.73 (2H, m), 3.21-3.44 (2H, m), 2.21-2.33(8H, m), 1.62-1.71 (2H, m), 1.29 (3H, s); MS (ES+) [M+H]⁺=364.2.

6.8. Example 8 Synthesis ofN-(3-(isopropylcarbamoyl)phenyl)-4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide

To a solution of4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4yl)-piperidine-4-carboxylicacid from example 6, step B, (0.030 g, 0.11 mmol) in DMF (1 mL) wereadded HATU (0.042 g, 0.11 mmol), triethylamine (0.028 mL, 0.22 mmol) and3-amino-N-isopropyl-benzamide (0.029 mg, 0.17 mmol). The reaction wasstirred at 50° C. for 48 hours and concentrated under vacuum. Theresidue was purified by prep HPLC to give the title compound (0.023 g,48%).

¹HNMR (CD₃OD): δ 8.19 (1H, s), 7.99 (1H, m), 7.7-7.72 (1H, m), 7.56-7.58(1H, m), 7.41-7.43 (1H, J=8 Hz and 15.6 Hz, dd), 7.00 (1H, J=0.8 Hz, d),4.20-4.23 (1H, m), 3.79-3.85 (2H, m), 3.36-3.46 (2H, m), 2.38-2.44 (5H,m), 1.76-1.83 (2H, m), 1.42 (3H, s), 1.268 (6H, J=6.8 Hz, d); MS (ES+)[M+H]⁺=435.

6.9. Example 9 Synthesis ofN-(3-tert-butylphenyl)-4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide

A. Preparation of tert-Butyl4-(3-tert-butylphenylcarbamoyl)-4-methylpiperidine-1-carboxylate. To asolution of 1-(tert-butoxycarbonyl)-4-methylpiperidine-4-carboxylic acid(0.30 g, 1.2 mmol), DMF (20 drops), and pyridine (0.38 g, 4.8 mmol) indichloroethane (10 mL) was added oxalyl chloride (0.15 g, 1.2 mmol). Themixture was stirred for 40 minutes, and 3-tert-butylaniline was added.The mixture was stirred for 1 hour, diluted with dichloromethane, washedtwice each with sat. aq. NaHCO₃ and 1.0 N aq. HCl, dried over MgSO₄, andconcentrated. Silica gel chromatography (0-2% MeOH:CH₂Cl₂) gave thetitle compound in 61 % yield. MS (ES+) [M+H]⁺=375.

B. Preparation ofN-(3-tert-butylphenyl)-4-methylpiperidine-4-carboxamide. tert-Butyl4-(3-tert-butylphenylcarbamoyl)-4-methylpiperidine-1-carboxylate fromstep A (0.4 g, 1.07 mmol) was treated with HCl (4.0 N in dioxane, 1.3mL, 5.34 mmol) in methanol (5.0 mL) at 50° C. for 30 minutes. Themixture was concentrated to give the title compound as the hydrochloridesalt in quantitative yield. MS (ES+) [M+H]⁺=275.

C.N-(3-tert-butylphenyl)-4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide.N-(3-tert-Butylphenyl)-4-methylpiperidine-4-carboxamide from step B(0.33 g, 1.07 mmol), 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (0.18g, 1.07 mmol), N,N-diisopropylethylamine (0.69 g. 5.34 mmol), andisopropanol (7 mL) were combined in a pressure rated sealed tube andheated at 100° C. for 18 hours. The mixture was concentrated and takenup in dichloromethane from which a precipitate (0.18 g) was collected.The solution was chromatographed (SiO₂, 0-2% MeOH:CH₂Cl₂), and thecolumned material (0.15 g) was combined with the precipitate anddissolved in methanol (15 mL) at 60° C. Water was added (5.0 mL), andthe mixture was cooled. The resultant precipitate was filtered, washedwith water, and dried to give the title compound in 50% yield.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.51 (s, 1H), 9.31 (s, 1H), 8.19 (s,1H), 7.62 (s, 1H), 7.59 (d, J=8.0 Hz, 1H), 7.22 (t, J=8Hz and 16Hz, 1H),7.08 (d, J=8.0 Hz, 1H), 3.63-3.68 (m, 2H), 3.28-3.33 (m, 2H), 2.36 (s,3H), 2.25-2.29 (m, 2H), 1.63-1.68 (m, 2H), 1.31 (s, 3H), 1.27 (S, 9H);MS (ES+) [M+H]⁺=406.

6.10. Example 10 Synthesis of4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-(3-(oxazol-5-yl)phenyl)piperidine-4-carboxamide

A. Preparation of tert-butyl4-methyl-4-(3-(oxazol-5-yl)phenylcarbamoyl)-piperidine-1-carboxylate. Toa solution of 1-(tert-butoxycarbonyl)-4-methylpiperidine-4-carboxylicacid (61.3 mg, 0.252 mmol) and pyridine (79.7 mg, 0.08 mL, 1.01 mmol) in1,2-dichloroethane (2 mL) at 0° C. was added DMF (1-2 drops) followed byoxalyl chloride (31.9 mg, 0.022 mL, 0.252 mmol). The reaction wasstirred for 0.5 hour, and 3-(oxazol-5-yl)aniline (40.4 mg, 0.252 mmol)was added. The reaction was allowed to stir for another 1.5 hours. Themixture was diluted with ethyl acetate (10 mL), and the organics werewashed with 0.1 N HCl (2×10 mL), brine (4 mL), dried over MgSO₄,filtered, and concentrated to give the title compound, which was used inthe next step without further purification. MS (ES+) [M+H]⁺=386.2.

B. Preparation of4-methyl-N-(3-(oxazol-5-yl)phenyl)piperidine-4-carboxamide. To asolution of tert-butyl4-methyl-4-(3-(oxazol-5-yl)phenylcarbamoyl)piperidine-1-carboxylate fromstep A (77.7 mg, 0.202 mmol) in methanol (3 mL) was added HCl (4.0 N indioxane, 0.4 mL, 1.6 mmol). The reaction was stirred for 15 hours andthen concentrated to dryness to give the title compound as thehydrochloride salt. MS (ES+) [M+H]⁺=286.2.

C. Preparation of4-methyl-1-(5-methyl-7H-pyrrolo[23-d]pyrimidin-4-yl)-N-(3-(oxazol-5-yl)phenyl)piperidine-4-carboxamide.A pressure vessel was charged with a solution of4-methyl-N-(3-(oxazol-5-yl)phenyl)piperidine-4-carboxamide hydrochloridefrom step B (51.9 mg, 0.17 mmol) and isopropanol (3 mL), and to thissolution was added 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (55.0mg, 0.329 mmol) and N,N-diisopropylethylamine (297.9 mg, 0.40 mL, 2.31mmol). The vessel was sealed, and the reaction was heated to 105° C. for15 hours. The reaction was worked up by normal means, and the residuewas purified by prep HPLC to afford the title compound as an off-whitesolid.

¹H NMR (400 MHz) δ ppm 8.27 (s, 1H), 8.18 (s, 1H), 8.03 (t, J=1.77 Hz,1H), 7.58 (ddd, J=8.08, 2.02, 1.01 Hz, 1H), 7.52 (s, 1H), 7.50 (dt,J=8.08, 1.26 Hz, 1H), 7.43 (t, J=7.83 Hz, 1H), 7.00 (app. d, J=0.76 Hz,1H), 3.78-3.86 (m, 2H), 3.38-3.46 (m, 2H), 2.44 (d, J=0.76 Hz, 3H),2.36-2.44 (m, 2H), 1.79 (ddd, J=13.39, 9.85, 3.54 Hz, 2H), 1.42 (s, 3H);MS (ES+) [M+H]⁺=417.2.

6.11. Example 11 Synthesis of4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-(3-sulfamoylphenyl)piperidine-4-carboxamide

A. Preparation of tert-butyl4-methyl-4-(3-sulfamoylphenylcarbamoyl)piperidine-1-carboxylate. To asolution of 1-(tert-butoxycarbonyl)-4-methylpiperidine-4-carboxylic acid(60 mg, 0.25 mmol) in dichloroethane (2.0 mL) were added DMF (2 drops)and pyridine (78 mg, 0.99 mmol), followed by oxalyl chloride (31 mg,0.25 mmol). The mixture was stirred for 40 minutes, and3-amino-benzenesulfonamide was added. The mixture was heated at 50° C.for 3 hours, and the mixture was cooled, diluted with dichloromethane,washed twice each with sat. aq. NaHCO₃ and 1.0 N aq. HCl, dried overMgSO₄, and concentrated. Silica gel chromatography (2-5% MeOH:CH₂Cl₂)gave the title compound in 40% yield. MS (ES+) [M+H]⁺=398.

B. Preparation of4-methyl-N-(3-sulfamoylphenyl)piperidine-4-carboxamide. tert-Butyl4-methyl-4-(3-sulfamoylphenylcarbamoyl)piperidine-1-carboxylate fromstep A (39 mg, 0.098 mmol) was treated with HCl (4.0 N in dioxane, 98μL, 0.39 mmol) in methanol (1.0 mL) at 50° C. for 45 minutes. Themixture was concentrated to give the title compound as the hydrochloridesalt in quantitative yield. MS (ES+) [M+H]⁺=298.

C. Preparation of4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-N-(3-sulfamoylphenyl)piperidine-4-carboxamide.4-Methyl-N-(3-sulfamoylphenyl)piperidine-4-carboxamide from step B (33mg, 0.098 mmol), 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (16 mg,0.098 mmol), N,N-diisopropylethylamine (51 mg, 0.39 mmol), andisopropanol (1.0 mL) were combined in a pressure rated sealed tube andheated at 105° C. for 18 hours. The mixture was concentrated, and theresidue was purified by preparative HPLC to give the title compound in21% yield.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.52 (s, 1H), 9.72 (s, 1H), 8.24 (s,1H), 8.19 (s, 1H), 7.87-7.90 (m, 1H), 7.50-7.52 (m, 2H), 7.35 (s, 2H),7.06 (s, 1H), 4.09-4.13 (m, 1H), 3.66-3.40 (m, 2H), 3.25-3.31 (m, 2H),3.18 (d, J=4 Hz, 2H), 2.36 (s, 3H), 2.27-2.30 (m, 2H), 1.64-1.70 (m,2H), 1.32 (s, 3H); MS (ES+) [M+H]⁺=429.

6.12. Example 12 Synthesis ofN-(3-(1H-tetrazol-5-yl)phenyl)-4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide

A. Preparation of tert-butyl4-(3-(1H-tetrazol-5-yl)phenylcarbamoyl)-4-methylpiperidine-1-carboxylate.To a solution of 1-(tert-butoxycarbonyl)-4-methylpiperidine-4-carboxylicacid (100 mg, 0.41 mmol) in dichloroethane (2.0 mL) were added DMF (2drops) and pyridine (0.13 g, 1.6 mmol), followed by oxalyl chloride (57mg, 0.45 mmol). The mixture was stirred for 40 minutes, and3-(1H-tetrazol-5-yl)-phenylamine was added. The mixture was stirred for2 hours, and a pink precipitate formed. The mixture was filtered, andthe filtrate was washed with 1.0 N aq. HCl and brine, dried over MgSO₄,and concentrated to give the title compound in 70% yield. MS (ES+)[M+H]⁺=387.

B. Preparation ofN-(3-(1H-tetrazol-5-yl)phenyl)-4-methylpiperidine-4-carboxamide.tert-Butyl4-(3-(1H-tetrazol-5-yl)phenylcarbamoyl)-4-methylpiperidine-1-carboxylatefrom step A (0.11 g, 0.29 mmol) was treated with HCl (4.0 N in dioxane,0.36 mL, 1.45 mmol) in methanol (2.0 mL) and heated at 50° C. for 30minutes. The mixture was concentrated to give the title compound as thehydrochloride salt in quantitative yield. MS (ES+) [M+H]⁺=287.

C. Preparation ofN-(3-(1H-tetrazol-5-yl)phenyl)-4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide.N-(3-( 1H-tetrazol-5-yl)phenyl)-4-methylpiperidine-4-carboxamide fromstep B (93 mg, 0.29 mmol), 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine(49 mg, 0.29 mmol), N,N-diisopropylethylamine (0.19 g. 1.45 mmol),isopropanol (4.0 mL), and DMSO (0.5 mL) were combined in a pressurerated sealed tube and heated at 105° C. for 18 hours. The mixture wasconcentrated, and the residue was purified by preprative HPLC to givethe title compound in 4.3% yield.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.5 (s, 1H), 9.56 (s, 1H), 8.31 (s,1H), 8.19 (s, 1H), 7.77 (d, J=8 Hz, 1H), 7.69 (d, J=8 Hz, 1H), 7.40 (t,J=8 and 16 Hz, 1H), 7.05 (s, 1H), 3.64-3.72 (m, 2H), 2.37 (s, 3H),2.29-2.33 (m, 2H), 1.91 (s, 2H), 1.64-1.70 (m, 2H), 1.34 (s, 3H); MS(ES+) [M+H]⁺=418.

6.13. Example 13 Synthesis of3-(4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of tert-butyl4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)-4-methylpiperidine-1-carboxylate.1-(tert-Butoxycarbonyl)-4-methylpiperidine-4-carboxylic acid (67 mg,0.28 mmol), EDC (64 mg, 0.33 mmol), HOBT (45 mg, 0.33 mmol) anddichloroethane (2.0 mL) were combined in a pressure rated sealed tubeand stirred for 45 min. 3-Aminophenyl dimethylcarbamate (50 mg, 0.28mmol) was added, and the mixture was heated at 85° C. for 18 hours. Themixture was concentrated and chromatographed (SiO₂, 0-2% MeOH:CH₂Cl₂) togive the title compound in 50% yield. MS (ES+) [M+H]⁺=406.

B. Preparation of 3-(4-methylpiperidine-4-carboxamido)phenyldimethylcarbamate. tert-Butyl4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)-4-methylpiperidine-1-carboxylatefrom step A (56 mgs, 0.138 mmol) was treated with HCl (4.0 N in dioxane,0.17 mL, 0.69 mmol) in methanol (2.0 mL) at 40° C. for 3 hours. Themixture was concentrated to give the title compound as the hydrochloridesalt in quantitative yield. MS (ES+) [M+H]⁺=306.

C. Preparation of3-(4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate. 3-(4-methylpiperidine-4-carboxamido)phenyldimethylcarbamate from step B (47 mg, 0. 14 mmol)4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (23 mg, 0.138 mmol),N,N-diisopropylethylamine (54 mg, 0.41 mmol), and isopropanol (1.0 mL)were combined in a pressure rated sealed tube and heated at 100° C. for18 hours. The mixture was concentrated, and the residue was purified bypreparative HPLC to give the title compound in 53% yield.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.18 (s, 1H), 7.50 (s, 1H), 7.42-7.44 (d,J=8 Hz, 1H), 7.34 (t, J=8 and 16 Hz, 1H), 7.0 (s, 1H), 6.87-6.89 (m,1H), 3.79-3.85 (m, 2H), 3.37-3.44 (m, 2H), 3.14 (s, 1H), 3.01 (s, 3H),2.44 (s, 3H), 2.36-2.40 (m, 2H), 1.75-1.81 (m, 2H), 1.40 (s, 3H); MS(ES+) [M+H]⁺=437.

6.14. Example 14 Synthesis of3-(4-benzyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of tert-butyl4-benzyl-4-(3-(dimethylcarbamoyloxy)-phenylcarbamoyl)piperidine-1-carboxylate.Under an atmosphere ofN₂,4-benzyl-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (0.2 g,0.626 mmol), pyridine (0.202 mL, 2.5 mmol), and DMF (3 drops) weredissolved in CH₂Cl₂ (10 mL) and cooled in an ice bath. To the cooledsolution, oxalyl chloride (0.06 mL, 0.688 mmol) was added dropwise. Thereaction was then removed from the ice bath and stirred at roomtemperature for 30 min. The reaction was re-cooled in an ice bath, and3-aminophenyl dimethylcarbamate (0.112 g, 0.626 mmol) was added in oneportion. The reaction was stirred overnight, and then washed with 1 Naq. HCl and brine, dried over MgSO₄, and concentrated under vacuum. Theresidue was purified by silica gel chromatography (ethyl acetate:hexanesgradient) to give the title compound in 54% yield. MS (ES+)[M+NH₄]⁺=500.

B. Preparation of 3-(4-benzylpiperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of tert-butyl4-benzyl-4-(3-(dimethylcarbamoyloxy)-phenylcarbamoyl)piperidine-1-carboxylatefrom step A (0.162 g, 0.336 mmol) in MeOH (5 mL) was added 4 M HCl indioxane ( 0.250 mL, 1.00 mmol). The reaction was heated at 50° C. for 1hour, and then concentrated under vacuum, re-dissolved in MeOH andre-concentrated twice to give the title compound as the hydrochloridesalt. MS (ES+) [M+H]⁺=382.

C. Preparation of3-(4-benzyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate. 3-(4-benzylpiperidine-4-carboxamido)phenyldimethylcarbamate (0.140 g, 0.336 mmol) from step B,4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (0.056 g, 0.336 mmol),N,N-diisopropylethylamine (0.175 mL, 1.00 mmol), and isopropanol (5 mL)were combined and heated at 120° C. overnight in a pressure vessel. Thereaction was diluted with CH₂Cl₂, washed with H₂O and brine, dried overMgSO₄, and concentrated under vacuum. The residue was taken up in hotethanol/H₂O. Upon cooling, the product precipitated out of solution togive the title compound in 81% yield.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.18 (s, 1H), 7.15-7.37 (m, 8H), 7.00 (s,1H), 6.88-6.91 (m, 1H), 3.97 (d, J=13.6 Hz, 2H), 3.25 (t, J=12,24 Hz,2H), 3.14 (s, 3H), 3.02 (s, 2H), 2.96 (s, 3H), 2.40 (s, 3H), 2.40 (d,J=14.4 Hz, 2H), 1.85-1.92 (m, 2H); MS (ES+) [M+H]⁺=513.

6.15. Example 15 Synthesis of3-(4-(benzyloxymethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of 1-tert-butyl 4-ethyl4-(benzyloxymethyl)piperidine-1,4-dicarboxylate. LDA (2.64 mL, 5.82mmol) was added to a solution of 1-tert-butyl 4-ethylpiperidine-1,4-dicarboxylate (1 g, 3.88 mmol) in THF (35 ML) at −78° C.The reaction was stirred for 30 min, and then BOMCl (0.6 g, 3.88 mmol)was added. The reaction was allowed to warm to room temperature, andthen quenched with aq. NH₄Cl. The aqueous layer was extracted withEtOAc, and the organic extract was washed with brine, dried over MgSO₄,and concentrated under vacuum. The residue was purified by flashchromatography (80 g SiO₂, 0-10% ethylacetate:hexanes) to afford thetitle compound (0.5 g, 34%). MS (ES+) [M+H]⁺=378.

B. Preparation of4-(benzyloxymethyl)-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid.1-tert-Butyl 4-ethyl 4-(benzyloxymethyl)piperidine-1,4-dicarboxylatefrom step A (0.500 g, 1.3 mmol) was dissolved in 1 N aq. LiOH (15 mL)and THF (10 mL) and was heated at 50° C. for 24 hours. The reactionmixture was acidified with 1 N HCl and extracted with Et₂O. The organicextract was washed with brine, dried over MgSO₄, and concentrated undervacuum to afford the title compound (0.3 g, 65%). MS (ES+) [M+H]⁺=349.

C. Preparation of tert-butyl4-(benzyloxymethyl)-4-(3-(dimethylcarbamoyloxy)-phenylcarbamoyl)piperidine-1-carboxylate.To a solution of4-(benzyloxymethyl)-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acidfrom step B (0.3 g, 0.85 mmol) in isopropyl acetate (10 mL) was addedN,N-diisopropylethylamine (0.165 mL, 0.94 mmol) and HATU (0.94 mmol).The reaction was heated at 85° C. for 30 min, and 3-aminophenyldimethylcarbamate was added to the mixture. The reaction was heated at85° C. for 2 hours, then worked up by standard procedures. The productwas isolated by flash chromatography (40 g SiO₂, 0-30%ethylacetate:hexanes) to give the title compound (0.25 g, 57%). MS (ES+)[M+H]⁺=512.

D. Preparation of 3-(4-(benzyloxymethyl)piperidine-4-carboxamido)phenyldimethylcarbamate. tert-Butyl4-(benzyloxymethyl)-4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)piperidine-1-carboxylatefrom step C (0.25 g, 0.49 mmol) was dissolved in isopropanol, treatedwith 4 N HCl, and stirred for two hours. The reaction mixture wasadjusted to pH 7-8 with NaHCO₃, and then extracted with Et₂O. Theorganic extracts were concentrated under vacuum to give the titlecompound (0.2 g, 100%). MS (ES+) [M+H]⁺=412.

E. Preparation of3-(4-(benzyloxymethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of3-(4-(benzyloxymethyl)piperidine-4-carboxamido)phenyl dimethylcarbamatefrom step D (0.2 g, 0.49 mmol) in isopropanol (5 mL) was addedN,N-diisopropylethylamine (0.167 mL, 0.98 mmol) and4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (0.08 g, 0.49 mmol). Thereaction was heated at 85° C. overnight, diluted with EtOAc, washed withbrine, dried over MgSO₄, and concentrated under vacuum. The residue waspurified by flash chromatography (40 g SiO₂, 0-80% ethylacetate:hexanes)to give the title compound (0.1 g, 38%).

¹H NMR (400 MHz, CD₃OD) δ ppm 10.68 (s, 1H), 8.78 (s, 1H), 8.37 (s, 1H),7.33-7.44 (m, 6H), 7.25 (t, J=8, 16 Hz, 1H), 7.11 (d, J=8 Hz, 1H), 6.91(s, 1H), 6.86 (d, J=8 Hz, 1H), 4. 62 (s, 2H), 3.65 (s, 2H), 3.62-3.75(m, 4 H), 3.09 (s, 3H), 3.01 (s, 3H), 2.40 (s, 3H), 2.27-2.33 (m, 2H),1.75-1.82 (m, 2H); MS (ES+) [M+H]⁺=543.

6.16. Example 16 Synthesis of3-(4-(hydroxymethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

To a solution of3-(4-(benzyloxymethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate from example 15, step E, (30 mg, 0.055 mmol) in THF (2mL) was added Pd/C (10%). The reaction mixture was stirred under H₂ at60 psi overnight. The mixture was filtered and concentrated undervacuum. The residue was purified by prep HPLC (30×250 mm C18 column,10-100% MeCN:H₂O (10 mM NH₄OAc), 18 min, 45 mL/min) to give the titlecompound (1 mg).

¹H NMR (400 MHz, CD₃OD) δ ppm 8.18 (s, 1H), 7.52 (s, 1H), 7.40-7.42 (m,1H), 7.33 (t, J=8, 16 Hz, 1H), 7.00 (s, 1H), 6.87 (d, J=8 Hz, 1H),3.84-3.89 (m, 2H), 3.75 (s, 2H), 3.32-3.44 (m, 2H), 3.14 (s, 3H), 3.00(s, 3H), 2.45 (s, 3H), 2.38 (d, J=12 Hz, 2H), 1.72-1.81 (m, 2H); MS(ES+) [M+H]⁺=453.

6.17. Example 17 Synthesis of3-(4-(methoxymethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate

A. Preparation of 1-tert-butyl 4-ethyl4-(methoxymethyl)piperidine-1,4-dicarboxylate. To a solution of1-tert-butyl 4-ethyl piperidine-1,4-dicarboxylate (1.0 g, 4.07 mmol) inTHF (20 mL) at −78° C. under nitrogen was added dropwise LDA (1.5 M inTHF, 4.0 mL, 6.10 mmol). This solution was stirred for 1 hour at −78°C., after which methoxymethyl chloride (0.8 mL, 10.5 mmol) was added.The system was allowed to warm slowly to room temperature over 2 hours.The reaction was quenched with aqueous NH₄Cl and extracted with ethylacetate (3×50 mL). The organic layer was dried over a pad of silica gel,and the solvent removed under vacuum. The title compound was isolated bysilica gel chromatography (5-10% ethyl acetate:hexanes) as a clear oil(830 mg, 69% yield). MS (ES+) [M+H]⁺=302.4.

B. Preparation of1-(tert-butoxycarbonyl)-4-(methoxymethyl)-piperidine-4-carboxylic acid.To a solution of 1-tert-butyl 4-ethyl4-(methoxymethyl)piperidine-1,4-dicarboxylate from step A (732 mg, 2.63mmol) in 1:2 ethanol/water (9 mL) was added lithium hydroxidemonohydrate (700 mg). The mixture was heated to 75° C. for 1 hour. Thereaction was cooled to room temperature and poured into 50 mL of sat.aq. NaHSO₄. The aqueous layer was extracted with ethyl acetate (3×50mL). The combined organic layers were washed with brine, dried overMgSO₄, and concentrated under vacuum to give the title compound as aviscous yellow oil, which was carried on to the next step withoutfurther purification. MS (ES+) [M+H]⁺=274.2.

C. Preparation of tert-butyl4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)-4-(methoxymethyl)piperidine-1-carboxylate.To a solution of1-(tert-butoxycarbonyl)-4-(methoxymethyl)-piperidine-4-carboxylic acidfrom step B (718 mg, 2.63 mmol), pyridine (0.84 mL, 10.52 mmol), and DMF(catalytic, 3 drops) in CH₂Cl₂ (13 mL) was added dropwise oxalylchloride (0.25 mL, 2.89 mmol, caution: exothermic). After gas evolutionsubsided, the reaction was stirred at ambient temperature for 30minutes. 3-Aminophenyl dimethylcarbamate (475 mg, 2.63 mmol) was addedin one portion to this solution and stirred for another 30 minutes. Thereaction was quenched with saturated aqueous NH₄Cl, and the aqueouslayer was extracted with ethyl acetate (3×50 mL). The combined organiclayers were washed with brine, dried over MgSO₄, filtered, andconcentrated under vacuum. Silica gel chromatography (50% ethylacetate:hexanes) afforded the title compound (1.03 g, 90% yield for twosteps). MS (ES+) [M+NH₄]⁺=453.3

D. Preparation of 3-(4-(methoxymethyl)piperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of tert-butyl4-(3-(dimethylcarbamoyloxy)-phenylcarbamoyl)-4-(methoxymethyl)piperidine-1-carboxylatefrom step C (1.03 g, 2.37 mmol) in methanol (10 mL) was added HCl (4 Msolution in dioxane, 2 mL). The reaction was heated at 50° C. for 1hour, and then concentrated under vacuum to provide the title compoundas the hydrochloride salt. MS (ES+) [M+H]⁻=336.3.

E. Preparation of3-(4-(methoxymethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of3-(4-(methoxymethyl)piperidine-4-carboxamido)phenyl dimethylcarbamate,from step D, and N,N-diisopropylethylamine (2 mL, 11.9 mmol) inisopropanol (5 mL) was added4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (397 mg, 2.37 mmol). Thereaction mixture was heated to 110° C. in a sealed high-pressure vialfor 17 hours, during which time the mixture became homogenous. Thereaction was concentrated under vacuum and purified by silica gelchromatography (3-5% methanol:CH₂Cl₂). The product was washed with coldmethanol to provide the title compound as an off-white solid (462 mg,42% yield).

¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.50 (br. s., 1H), 9.45 (s, 1H), 8.18(s, 1H), 7.54-7.57 (m, 1H), 7.47 (d, J=8.84 Hz, 1H), 7.29 (t, J=8.08 Hz,1H), 7.04 (s, 1H), 6.81 (dd, J=8.08, 2.27 Hz, 1H), 3.68-3.78 (m, 2H),3.56 (s, 2H), 3.26 (s, 3H), 3.21 (m, 2H), 3.04 (s, 3H), 2.91 (s, 3H),2.34 (s, 3H), 2.22-2.31 (m, 2H), 1.65-1.78 (m, 2H); MS (ES+)[M+H]⁺=467.3.

6.18. Example 18 Synthesis of3-(4-((2-methoxyethoxy)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of 1-tert-butyl 4-ethyl4-((2-methoxyethoxy)methyl)piperidine-1,4-dicarboxylate. To a solutionof 1-tert-butyl 4-ethyl piperidine-1,4-dicarboxylate (2.0 g, 7.8 mmol)in THF (70 mL) at −78° C. was added dropwise LDA over 8 minutes,maintaining the temperature below −75° C. The mixture was stirred at−78° C. for 1 hour. 1-Chloromethoxy-2-methoxy-ethane (0.97 g. 7.8 mmol)was added dropwise while maintaining the temperature below −75° C. Themixture was stirred at −78° C. for 3 hours, and then allowed to warmslowly to room temperature overnight. The mixture was quenched with sat.aq. NH₄Cl and extracted 3 times with EtOAc. The organics were combined,washed with brine, dried over MgSO₄, and concentrated. Silica gelchromatography (10-20% ethyl acetate:hexanes) gave the title compound in67% yield. MS (ES+) [M+H]⁺=346.

B. Preparation of1-(tert-butoxycarbonyl)-4-(2-methoxyethoxy)piperidine-4-carboxylic acid.1-tert-Butyl 4-ethyl4-((2-methoxyethoxy)methyl)piperidine-1,4-dicarboxylate from step A(1.78 g, 5.15 mmol) was treated with LiOH (0.62 g. 26 mmol) in ethanol(12 mL) and water (24 mL) at 80° C. for 18 hours. The mixture wascooled, diluted with dichloromethane, and washed with sat. aq. NaHSO₄.The aqueous phase was back extracted with dichloromethane (3×), and theorganics were combined, washed with brine, dried over MgSO₄, andconcentrated to give the title compound in quantitative yield. MS (ES+)[M+H]⁺=318.

C. Preparation of tert-butyl4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)-4-(2-methoxyethoxy)piperidine-1-carboxylate.To a solution of1-(tert-butoxycarbonyl)-4-(2-methoxyethoxy)piperidine-4-carboxylic acidfrom step B (1.6 g, 5.1 mmol) in dichloroethane was added pyridine (1.6g, 20 mmol) and DMF (0.05 mL), followed by oxalyl chloride (0.65 g, 5.1mmol). The mixture was stirred for 30 minutes, and then 3-aminophenyldimethylcarbamate (0.91 g, 5.04 mmol) was added. The mixture was stirredfor 18 hours, then diluted with dichloromethane, washed twice with 1 Naq. HCl and once with brine, dried over MgSO₄, and concentrated. Silicagel chromatography (0-5% MeOH:CH₂Cl₂) gave the title compound in 88%yield. MS (ES+) [M+H]⁺=480.

D. Preparation of 3-(4-(2-methoxyethoxy)piperidine-4-carboxamido)phenyldimethylcarbamate. tert-Butyl4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)-4-(2-methoxyethoxy)piperidine-1-carboxylatefrom step C (2.1 g, 4.4 mmol) was treated with HCl (4.0 N in dioxane,5.5 mL, 22 mmol) in methanol (20 mL) at 50° C. for 30 minutes. Themixture was concentrated to give the title compound as the hydrochloridesalt in quantitative yield. MS (ES+) [M+H]⁺=380.

E. Preparation of3-(4-((2-methoxyethoxy)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate. 3-(4-(2-Methoxyethoxy)piperidine-4-carboxamido)phenyldimethylcarbamate from step D (1.82 g, 4.38 mmol),4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (0.73 g, 4.4 mmol),N,N-diisopropylethylamine (2.8 g. 22 mmol), and isopropanol (50 mL) werecombined in a pressure rated sealed tube and heated at 115° C. for 18hours. The mixture was concentrated and chromatographed (SiO₂, 0-5%MeOH:CH₂Cl₂) to give the title compound in 61% yield.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 11.48 (s, 1H), 9.43 (s, 1H), 8.18 (s,1H), 7.52 (s, 1H), 7.44 (d, J=8 Hz, 1H), 7.30 (t, J=8 and 16 Hz, 1H),7.05 (s, 1H), 6.81 (d, J=8 Hz, 1H), 3.68-3.76 (m, 2H), 3.67 (s, 2H),3.55-3.58 (m, 2H), 3.44-3.47 (m, 2H), 3.24-3.30 (m, 2H), 3.23 (s, 3H),3.04 (s, 3H), 2.91 (s, 3H), 2.35 (s, 3H), 2.26 (d, J=16 Hz, 2H),1.69-1.76 (m, 2H); MS (ES+) [M+H]⁺=511.

6.19. Example 19 Synthesis of3-(4-((benzyloxycarbonylamino)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of 1-tert-butyl 4-methyl4-((benzyloxycarbonylamino)-methyl)piperidine-1,4-dicarboxylate. To asolution of 1-tert-butyl 4-methyl4-(aminomethyl)piperidine-1,4-dicarboxylate (0.300 g, 1.1 mmol) andN,N-diisopropylethylamine (0.229 mL, 1.32 mmol) in CH₂Cl₂ (5 mL) at 0°C. was added benzyl chloroformate. The reaction was stirred overnight,and then diluted with CH₂Cl₂ and washed with H₂O and brine, dried overMgSO₄, and concentrated under vacuum. The residue was purified by silicagel chromatography (EtOAc:hexanes gradient) to give the title compoundin 82% yield. MS (ES+) [M+H]⁺=407.

B. Preparation of4-((benzyloxycarbonylamino)methyl)-1-(tert-butoxycarbonyl)piperidine-4-carboxylicacid. To a solution of 1-tert-butyl 4-methyl4-((benzyloxycarbonylamino)methyl)piperidine-1,4-dicarboxylate from stepA in methanol (5 mL) was added 1 N aq. NaOH (0.615 mL, 0.615 mmol). Thereaction was heated at 50° C. overnight, acidified to pH 5 with 1 N aq.HCl, and extracted 3× with CH₂Cl₂. The combined organic extracts werewashed with brine, dried over Na₂SO₄, and concentrated under vacuum togive the title compound, which was carried on crude. MS (ES+)[M+H]⁺=393.

C. Preparation of tert-butyl4-((benzyloxycarbonylamino)methyl)-4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)piperidine-1-carboxylate.Under an atmosphere ofN₂,4-((benzyloxycarbonylamino)methyl)-1-(tert-butoxycarbonyl)piperidine-4-carboxylicacid (0.240 g, 0.611 mmol) from step B, pyridine (0.197 mL, 2.44 mmol)and DMF (3 drops) were dissolved in CH₂Cl₂ (10 mL) and cooled in an icebath. Oxalyl chloride (0.064 mL, 0.733 mmol) was added dropwise to thesolution. The reaction was then removed from the ice bath and stirred atroom temperature for 30 minutes. The reaction was re-cooled in an icebath, and 3-aminophenyl dimethylcarbamate (0.110 g, 0.611 mmol) wasadded in one portion. The reaction was stirred overnight, and thenwashed 1 N aq. HCl and brine, dried over MgSO₄, and concentrated undervacuum. The residue was purified by silica gel chromatography (ethylacetate:hexanes gradient) to give the title compound in 72% yield. MS(ES+) [M+NH₄]⁺=573.

D. Preparation of3-(4-((benzyloxycarbonylamino)methyl)piperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of tert-butyl4-((benzyloxycarbonylamino)methyl)-4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)-piperidine-1-carboxylatefrom step C (0.245 g, 0.441 mmol) in MeOH (2 mL) was added 4 M HCl indioxane (0.331 mL, 1.32 mmol). The reaction was heated at 50° C. for 1hour, and then concentrated under vacuum, re-dissolved in MeOH andre-concentrated twice to give the title compound as the hydrochloridesalt. MS (ES+) [M+H]⁺=455.

E. Preparation of3-(4-((benzyloxycarbonylamino)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate.3-(4-((Benzyloxycarbonylamino)methyl)piperidine-4-carboxamido)phenyldimethylcarbamate (0.216 g, 0.441 mmol) from step D,4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (0.073 g, 0.441 mmol),N,N-diisopropylethylamine (0.230 mL, 1.32 mmol), and isopropanol (5 mL)were combined and heated at 120° C. overnight in a pressure vessel. Thecrude reaction was diluted with CH₂Cl₂ and washed with H₂O and brine,dried over MgSO₄, and concentrated under vacuum. The residue waspurified by silica gel chromatography (MeOH:CH₂Cl₂ gradient) to give thetitle compound in 54% yield.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.18 (s, 1H), 7.49 (s, 1H), 7.40 (d, J=8Hz, 1H), 7.26-7.34 (m, 6H), 7.00 (s, 1H), 6.89 (d, J=8 Hz, 1H), 5.06 (s,2H), 3.92 (d, J=13.2 Hz, 2H), 3.50 (s, 2H), 3.28-3.37 (m, 2H), 3.1 (s,3H), 2.99 (s, 3H), 2.43 (s, 3H), 2.40 (d, J=12 Hz, 2H), 1.79 (t, J=10.6,21.2 Hz, 2H); MS (ES+) [M+H]⁺=586.

6.20. Example 20 Synthesis of3-(4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

3-(4-((Benzyloxycarbonylamino)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate (0.140 g, 0.239 mmol) from example 19, step E, Pd/C(10% Pd/C, Pearlman), and methanol (5 mL) were combined and hydrogenatedovernight at 1 atmosphere. The reaction was filtered through celite,concentrated under vacuum, and purified by neutral phase prep-HPLC togive the title compound as the acetate salt in 52% yield.

¹H NMR (400 MHz, D₂O) δ ppm 7.90 (s, 1H), 7.30 (t, J=8, 6 Hz, 1H),7.18-7.19 (m, 2H), 6.84-6.88 (m, 1H), 3.48 (d, J=13.6 Hz, 2H), 3.20 (s,2H), 3.08 (t, J=10.8, 21.6 Hz, 2H), 2.96 (s, 3H), 2.83 (s, 3H), 2.27 (d,J=14.4 Hz, 2H), 2.11 (s, 3H), 1.72 (t, J=10, 20 Hz, 2H); MS (ES+)[M+H]⁺=452.

6.21. Example 21 Synthesis of3-(4-((dimethylamino)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

To a solution of the acetate salt of3-(4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate from example 20 (14 mg, 0.027 mmol), acetic acid (6.2μl, 0.11 mmol), and NaBH₃CN (2.1 mg, 0.034 mmol) was added formaldehyde(37% in H₂O, 48 μl, 0.064 mmol). The reaction was stirred overnight,then concentrated, and the residue was purified by prep HPLC (30×100 mmC18 column, 5-70% MeCN:H₂O (10 mM NH₄OAc), 12 min, 45 mL/min.) andlyophilized to give the title compound (8 mg, 0.014 mmol, 57%) as theacetate salt.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.18 (s, 1 H), 7.51 (t, J=2.02 Hz, 1 H),7.37-7.42 (m, 1 H), 7.34 (t, J=8.08 Hz, 1 H), 7.00 (s, 1 H), 6.85-6.91(m, 1 H), 3.89 9d, J=13.64 Hz, 2 H), 3.38-3.48 (m, 2 H), 3.14 (s, 3 H),3.01 (s, 3 H), 2.72 (s, 2 H), 2.36-2.45 (m, 11 H), 1.79 (ddd, J=13.71,10.42, 3.41 Hz, 2 H); MS (ES+) [M+H]⁺=480.

6.22. Example 22 Synthesis of3-(4-(2-(dimethylamino)ethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of 1-tert-butyl 4-ethyl4-(2-(dimethylamino)ethyl)piperidine-1,4-dicarboxylate. To a solution of1-tert-butyl 4-ethyl piperidine-1,4-dicarboxylate (514 mg, 2 mmol) inTHF (10 mL) at −78° C. was added dropwise LDA (1.5 M in cyclohexane, 2mL, 3 mmol). After stirring for 30 minutes,2-bromo-N,N-dimethylethanamine (370 mg, 2.4 mmol) in THF (3 mL) wasadded. The reaction was stirred for 30 min at −78° C., then allowed towarm to room temperature, stirred for 2 hours, then quenched with sat.aq. NaHCO₃, diluted with EtOAc, washed with sat. aq. NaHCO₃ and brine(with back extraction), dried over MgSO₄, and concentrated under vacuum.The residue was purified by flash chromatography (40 g SiO₂, 0-15%MeOH:CH₂Cl₂) to give the title compound (328 mg, 1.0 mmol, 50%) as ayellow oil. MS (ES+) [M+H]⁺=329.

B. Preparation of1-(tert-butoxycarbonyl)-4-(2-(dimethylamino)ethyl)piperidine-4-carboxylicacid. 1-tert-Butyl 4-ethyl4-(2-(dimethylamino)ethyl)piperidine-1,4-dicarboxylate from step A (328mg, 1.0 mmol) was treated overnight with lithium hydroxide monohydrate(210 mg, 5 mmol) in 1:1 EtOH:H₂O (5 mL) at 60° C. The reaction wascooled to room temperature, neutralized to pH 6 with 1 N aq. HCl, andconcentrated under vacuum. The residue was purified by prep HPLC andlyophilized twice to give the title compound (191 mg, 64%) as a whitesolid. MS (ES+) [M+H]⁺=301.

C. Preparation of tert-butyl4-(2-(dimethylamino)ethyl)-4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)piperidine-1-carboxylate.1-(tert-Butoxycarbonyl)-4-(2-(dimethylamino)ethyl)piperidine-4-carboxylicacid from step B (100 mg, 0.33 mmol), HATU (175 mg, 0.46 mmol), andN,N-diisopropylethylamine (0.116 mL, 0.67 mmol) were stirred inisopropyl acetate for 10 minutes. 3-aminophenyl dimethylcarbamate (90mg, 0.5 mmol) was added, and the reaction was stirred at 85° C. for 2hours, cooled to room temperature, stirred overnight, and then dilutedwith EtOAc, washed with sat. aq. Na₂CO₃ and brine (with backextraction), dried over MgSO₄, filtered, and concentrated under vacuum,to give the title compound as a viscous oil. MS (ES+) [M+H]⁺=463.

D. Preparation of3-(4-(2-(dimethylamino)ethyl)piperidine-4-carboxamido)phenyldimethylcarbamate. Crude tert-butyl4-(2-(dimethylamino)ethyl)-4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)piperidine-1-carboxylatefrom step C was treated with 1:1 TFA:CH₂Cl₂ (1.5 mL) for 45 min. Thereaction was concentrated under vacuum, and the residue was purified byprep HPLC (30×100 mm C18 column, 0-50% MeOH:H₂O (0.1% TFA), 12 min., 45mL/min.) and lyophized to give impure title compound (173 mg) as the TFAsalt. MS (ES+) [M+H]⁺=363.

E. Preparation of3-(4-(2-(dimethylamino)ethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate. Impure3-(4-(2-(dimethylamino)ethyl)piperidine-4-carboxamido)phenyldimethylcarbamate from step D (173 mg) was combined with4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (50 mg, 0.30 mmol) andN,N-diisopropylethylamine (0.174 mL, 5 mmol) in isobutanol (0.4 mL) andheated at 110° C. for 7 hours. The reaction was cooled to roomtemperature and concentrated under vacuum. The residue was purified byprep HPLC (30×100 mm C18 column, 10-70% MeCN:H₂O (10 mM NH₄OAc), 12 min,45 mL/min) and lyophilized to give the title compound (60 mg) as thedi-acetate salt.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.19 (s, 1 H), 7.52 (t, J=2.15 Hz, 1 H),7.44 (d, J=8.08 Hz, 1 H), 7.35 (t, J=8.08 Hz, 1 H), 7.01 (s, 1 H), 6.91(dd, J=7.58, 1.77 Hz, 1 H), 3.91 (d, J=13.64 Hz, 2 H), 3.37-3.30 (m, 2H), 3.14 (s, 3 H), 3.01 (s, 3 H), 2.75-2.82 (m, 2 H), 2.58 (s, 6 H),2.43-2.47 (m, 5 H), 2.00-2.08 (m, 2 H), 1.79-1.89 (m, 2 H); MS (ES+)[M+H]⁺=494.

6.23. Example 23 Synthesis of3-(4-((1H-imidazol-4-yl)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of 1-tert-butyl 4-ethyl4-((1-trityl-1H-imidazol-4-yl)methyl)piperidine-1,4-dicarboxylate. To asolution of 1-tert-butyl 4-ethyl piperidine-1,4-dicarboxylate (360 mg,1.39 mmol) in anhydrous THF (10 mL) at −78° C. was added LDA (1.7 M inTHF, 1.07 mL, 1.8 mmol) dropwise over 10 min. The reaction mixture wasstirred at −78° C. for 1 hour, and4-(chloromethyl)-1-trityl-1H-imidazole (500 mg, 13.9 mmol) in THF (5 mL)was added slowly. The reaction mixture was allowed to warm to roomtemperature over 1 hour, quenched with sat. aq. NH₄Cl (10 mL), andextracted with EtOAc (3×20 mL). The combined organic extracts werewashed with brine, dried over Na₂SO₄, filtered, and concentrated undervacuum. Purification by silica gel chromatography (10-50% EtOAc:Hexanes)afforded the title compound (403 mg, 51%). MS (ES+) [M+H]⁺=580.3.

B. Preparation of1-(tert-butoxycarbonyl)-4-((1-trityl-1H-imidazol-4-yl)methyl)piperidine-4-carboxylicacid. To a solution of 1-tert-butyl 4-ethyl4-((1-trityl-1H-imidazol-4-yl)methyl)piperidine-1,4-dicarboxylate fromstep A (330 mg, 0.57 mmol) in 1:1:2 THF:MeOH:H₂O (10 mL) was added LiOH(136 mg, 5.7 mmol). The mixture was refluxed until LCMS showed completeconversion to product, then concentrated under vacuum, dissolved inwater (3 mL), neutralized to pH 5-6 with aq. NaHSO₄, and extracted withethyl acetate (3×10 mL). The combined organic phases were washed withbrine, dried over Na₂SO₄, and concentrated under vacuum to give thetitle compound (273 mg, 86%). MS (ES+) [M+H]⁺=552.2.

C. Preparation of tert-butyl4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)-4-((1-trityl-1H-imidazol-4-yl)methyl)piperidine-1-carboxylate.To a solution of1-(tert-butoxycarbonyl)-4-((1-trityl-1H-imidazol-4-yl)methyl)piperidine-4-carboxylicacid from step B (270 mg, 0.6 mmol) in isopropyl acetate (5 mL) wereadded HATU (273 mg, 0.73 mmol) and N,N-diisopropylethylamine (205 μL,1.2 mmol). The mixture was stirred for 5 min, and 3-aminophenyldimethylcarbamate (108 mg, 0.6 mmol) was added. The resulting mixturewas heated at 90° C. for 2 hours. The reaction mixture was diluted withEtOAc (10 mL), washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄,filtered, and concentrated under vacuum. The residue was purified bysilica gel chromatography (10-70% EtOAc:Hexanes) to afford the titlecompound (185 mg, 43%). MS (ES+) [M+H]⁺=714.2.

D. Preparation of3-(4-((1H-imidazol-4-yl)methyl)piperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of tert-butyl4-(3-(dimethylcarbamoyloxy)-phenylcarbamoyl)-4-((1-trityl-1H-imidazol-4-yl)methyl)piperidine-1-carboxylatefrom step C (180 mg) in MeOH (5 mL) was added HCl (1 N in dioxane, 1mL). The reaction was heated at 60° C. for 1 hour, and then concentratedunder vacuum. The residue was dissolved in EtOAc (5 mL), washed with aq.NaHCO₃ and brine, dried over Na₂SO₄, filtered, and concentrated undervacuum to produce the title compound (84 mg, 93%). MS (ES+)[M+H]⁺=372.2.

E. Preparation of3-(4-((1H-imidazol-4-yl)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate.3-(4-((1H-imidazol-4-yl)methyl)piperidine-4-carboxamido)phenyldimethylcarbamate from step D (10 mg, 0.027 mmol),4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (5 mg, 0.03 mmol), andN,N-diisopropylethylamine (23 μL, 0.135 mmol) were combined inisopropanol (0.5 mL). The resulting mixture was heated at 100° C. in asealed pressure tube for 24 hours. The reaction mixture was cooled andconcentrated under vacuum. The residue was purified by prep HPLC (10-95%MeOH:H₂O with 0.1% HCOOH), then treated with AcOH in H₂O and lyophilizedto give the title compound (5.7 mg, 42%) as the acetate salt.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.17 (s, 1 H), 7.61 (s, 1 H), 7.42-7.45(m, 1 H), 7.31-7.39 (m, 2 H), 6.99-7.01 (m, 1 H), 6.87-6.91 (m, 1 H),6.79 (s, 1 H), 3.90-3.98 (m, 2 H), 3.14 (s, 3 H), 3.02 (br. s., 2 H),3.01 (s, 3 H), 2.44 (d, J=1.01 Hz, 3 H), 2.36-2.43 (m, 2 H), 1.94 (s, 3H), 1.83-1.93 (m, 4 H); MS (ES+) [M+H]⁺=503.3.

6.24. Example 24 Synthesis of3-(4-(benzyloxy)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of 1-tert-butyl 4-methyl4-hydroxypiperidine-1,4-dicarboxylate. To a solution of methyl4-hydroxypiperidine-4-carboxylate (prepared as described in U.S. patentapplication no. 2005/0080095 to Zheng et al., filed on Apr. 14, 2005)(792 mg, 4.98 mmol) and triethylamine (1.04 mL, 7.47 mmol) indichloromethane (25 mL) was added di-tert-butyl dicarbonate (1.26 mL,5.48 mmol). Reaction progress was monitored by TLC analysis/ELSD. Uponcompletion, volatiles were removed under vacuum. Silica gelchromatography (0-10% MeOH:CH₂Cl₂) provided the title compound as aclear oil (1.25 g, 99% yield). MS (ES+) [M+H]⁺=250.2.

B. Preparation of 1-tert-butyl 4-methyl4-(benzyloxy)piperidine-1,4-dicarboxylate. To a solution of 1-tert-butyl4-methyl 4-hydroxypiperidine-1,4-dicarboxylate from step A (535 mg, 2.06mmol) in DMF (8.2 mL) was carefully added sodium hydride (60% dispersionin mineral oil, 124 mg, 3.09 mmol). The mixture was stirred for 30minutes at room temperature. Benzyl bromide (0.29 mL, 2.47 mmol) wasadded, and the reaction was stirred for another 30 minutes. Uponcompletion as monitored by TLC analysis, the reaction was quenched with50 mL of 1:1 saturated aq. NH₄Cl:water. The aqueous layer was extractedwith ethyl acetate (3×50 mL). The combined organic layers were washedwith brine, dried with MgSO₄, and concentrated under vacuum. Silica gelchromatography (5-10% ethyl acetate:hexanes) provided the title compoundas a clear oil (359 mg, 54% yield). MS (ES+) [M+H]⁺=340.3.

C. Preparation of1-(tert-butoxycarbonyl)-4-(benzyloxy)-piperidine-4-carboxylic acid. To asolution of 1-tert-butyl 4-methyl4-(benzyloxy)piperidine-1,4-dicarboxylate from step B (350 mg, 1.00mmol) in 1:2 methanol/water (6 mL) was added lithium hydroxidemonohydrate (200 mg). The mixture was heated to 60° C. for 30 min, andthen cooled to room temperature and poured onto saturated aqueous NaHSO₄(25 mL). The aqueous layer was extracted with ethyl acetate (3×25 mL).The combined organic layers were washed with brine, dried with MgSO₄,and concentrated under vacuum to give the title compound as a viscousoil, which was carried on to the next step without further purification.

D. Preparation of tert-butyl4-(benzyloxy)-4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)piperidine-1-carboxylate.To a solution of1-(tert-butoxycarbonyl)-4-(benzyloxy)-piperidine-4-carboxylic acid fromstep C (335 mg, 1.00 mmol), pyridine (0.43 mL, 5.40 mmol), and DMF(catalytic, 3 drops) in CH₂Cl₂ (6.8 mL) was added dropwise oxalylchloride (0.13 mL, 1.49 mmol, caution: exothermic). After gas evolutionsubsided, the reaction was stirred at ambient temperature for 30minutes. 3-Aminophenyl dimethylcarbamate (244 mg, 1.35 mmol) was addedin one portion to this solution, which was stirred for another 30minutes. The reaction was quenched with saturated aqueous NH₄Cl, and theaqueous layer was extracted with ethyl acetate (3×50 mL). The combinedorganic layers were washed with brine, dried with MgSO₄, filtered, andconcentrated under vacuum. Silica gel chromatography (40% ethylacetate:hexanes) afforded the title compound as a white solid (406 mg,82% yield. MS (ES+) [M+NH₄]⁺=515.4.

E. Preparation of 3-(4-(benzyloxy)piperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of tert-butyl4-(benzyloxy)-4-(3-(dimethylcarbamoyloxy)-phenylcarbamoyl)piperidine-1-carboxylatefrom step D (400 mg, 0.804 mmol) in methanol (4 mL) was added HCl (4 Msolution in dioxane, 0.8 mL). The reaction was heated at 50° C. for 1hour, and then concentrated under vacuum to provide the title compoundas the hydrochloride salt. MS (ES+) [M+H]⁺=398.4.

F. Preparation of3-(4-(benzyloxy)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of3-(4-(benzyloxy)piperidine-4-carboxamido)phenyl dimethylcarbamate, fromstep E, and N,N-diisopropylethylamine (0.7 mL, 4.02 mmol) in isopropanol(1.6 mL) was added 4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (135mg, 0.804 mmol). The reaction mixture was heated to 110° C. in a sealedhigh-pressure vial for 17 hours, during which time the mixture becamehomogenous. The reaction was concentrated under vacuum, and half of thematerial was purified by prep HPLC (30×100 mm C18 column, 20-100%acetonitrile:water (10 mM ammonium acetate), 15 min, 45 mL/min) toafford the title compound (33.4 mg) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ ppm 9.71-9.74 (br. s., 1H), 8.22 (s, 1H), 7.51(app q, J=1.85 Hz, 1H), 7.44-7.49 (m, 2H), 7.29-7.42 (m, 5H), 7.03 (d,J=1.01 Hz, 1H), 6.89 (ddd, J=7.83, 2.27, 1.26 Hz, 1H), 4.54 (s, 2H),3.91-3.99 (m, 2H), 3.45-3.54 (m, 2H), 3.14 (s, 3H), 3.01 (s, 3H), 2.46(d, J=1.01 Hz, 3H), 2.22-2.36 (m, 4H); MS (ES+) [M+H]⁺=529.3.

6.25. Example 25 Synthesis of3-(4-hydroxy-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

Crude3-(4-(benzyloxy)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate from example 24, step F (85% pure, approximately 80mg) was taken up in methanol (2 mL), and a catalytic amount ofPearlman's catalyst was added. The system was hydrogenated at 60 psi for24 hours, after which a small amount of product was observed.Trifluoroacetic acid (0.1 mL) was added, and the hydrogenation wascontinued at 60 psi H₂ for 3 days, after which the reaction wasapproximately one third of the way completed. The reaction was filtered,and the product was isolated by prep HPLC (30×100 mm C18 column, 10-100%acetonitrile:water (10 mM ammonium acetate), 15 min, 45 mL/min) toafford the title compound (9.6 mg) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.20 (s, 1H), 7.55 (t, J=2.02 Hz, 1H),7.44 (ddd, J=8.08, 2.02, 1.01 Hz, 1H), 7.33 (t, J=8.08 Hz, 1H), 6.99(app. d., J=1.01 Hz, 1H), 6.87 (ddd, J=8.08, 2.27, 0.76 Hz, 1H),3.95-4.03 (m, 2H), 3.73 (s, 3H), 3.41 (dt, J=12.63, 2.27 Hz, 2H),3.13-3.26 (m, 2H), 3.12 (s, 3H), 2.99 (s, 3H), 2.44 (d, J=0.76 Hz, 3H),2.36 (dt, J=13.39, 4.29 Hz, 2H), 1.75-1.83 (m, 2H). MS (ES+)[M+H]⁺=439.3.

6.26. Example 26 Synthesis of3-(4-(2-methoxyethoxy)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of 1-tert-butyl 4-methyl4-(2-methoxyethoxy)piperidine-1,4-dicarboxylate. To a solution of1-tert-butyl 4-methyl 4-hydroxypiperidine-1,4-dicarboxylate (360 mg,1.39 mmol) in DMF (5.6 mL) was carefully added sodium hydride (60%dispersion in mineral oil, 84 mg, 2.08 mmol). The mixture was stirredfor 30 minutes at room temperature. 1-Chloro-2-methoxyethane (0.38 mL,4.17 mmol) was added, and the reaction was stirred at 60° C. for 72hours. The reaction was quenched with 25 mL of water. The aqueous layerwas extracted with ethyl acetate (3×25 mL). The combined organic layerswere washed with brine, dried with MgSO₄, and concentrated under vacuum.Silica gel chromatography (20-35% ethyl acetate:hexanes) provided thetitle compound and starting material as a mixture that was carriedforward (150 mg, 1:2 product:starting material).

B. Preparation of1-(tert-butoxycarbonyl)-4-(2-methoxyethoxy)piperidine-4-carboxylic acid.The product mixture from step A (150 mg) was taken up in 1:2methanol:water (3 mL). Lithium hydroxide monohydrate (150 mg) was added,and the mixture was heated to 60° C. for 2 hours. The reaction wascooled to room temperature and poured onto saturated aqueous NaHSO₄ (25mL). The aqueous layer was extracted with ethyl acetate (3×25 mL). Thecombined organic layers were washed with brine, dried with MgSO₄, andconcentrated under vacuum to give a 2:1 mixture of1-(tert-butoxycarbonyl)-4-hydroxypiperidine-4-carboxylic acid and1-(tert-butoxycarbonyl)-4-(2-methoxyethoxy)piperidine-4-carboxylic acid,respectively, as a viscous oil, which was carried on to the next stepwithout further purification. MS (ES+) [M+H]⁺=304.2.

C. Preparation of tert-butyl4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)-4-(2-methoxyethoxy)piperidine-1-carboxylate.To a solution of carboxylic acids from step B, pyridine (0.2 mL, 2.48mmol), and DMF (catalytic, 2 drops) in CH₂Cl₂ (3.1 mL) was addeddropwise oxalyl chloride (60 μL, 0.68 mmol, caution: exothermic). Aftergas evolution subsided, the reaction was stirred at ambient temperaturefor 30 minutes. 3-Aminophenyl dimethylcarbamate (112 mg, 0.62 mmol) wasadded in one portion to this solution, which was stirred for another 30minutes. The reaction was quenched with saturated aq. NH₄Cl, and theaqueous layer was extracted with ethyl acetate (3×50 mL). The combinedorganic layers were washed with brine, dried with MgSO₄, filtered, andconcentrated under vacuum. Silica gel chromatography (80% ethylacetate:hexanes) afforded the title compound as an off-white solid (150mg). MS (ES+) [M+CH₃CN]⁺=529.3.

D. Preparation of 3-(4-(2-methoxyethoxy)piperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of tert-butyl4-(3-(dimethylcarbamoyloxy)-phenylcarbamoyl)-4-(2-methoxyethoxy)-piperidine-1-carboxylatefrom step C (150 mg, 0.322 mmol) in methanol (2 mL) was added HCl (4 Msolution in dioxane, 0.3 mL). The reaction was heated at 50° C. for 1hour, and then concentrated under vacuum to provide the title compoundas the hydrochloride salt. MS (ES+) [M+H]⁺=366.3.

E. Preparation of3-(4-(2-methoxylethoxy)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of3-(4-(2-methoxyethoxy)piperidine-4-carboxamido)phenyl dimethylcarbamatefrom step D, and N,N-diisopropylethylamine (0.28 mL, 1.6 mmol) inisopropanol (1.0 mL) was added4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (55 mg, 0.322 mmol). Thereaction mixture was heated to 110° C. in a sealed high-pressure vialfor 17 h, during which time the mixture became homogenous. The mixturewas concentrated under vacuum and purified by prep HPLC (30×100 mm C18column, 5-100% acetonitrile:water (10 mM NH₄OAc), 15 min, 45 mL/min) toafford the title compound (17.4 mg) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.20 (s, 1H), 7.51 (t, J=2.02 Hz, 1H),7.40 (ddd, J=8.08, 2.02, 1.01 Hz, 1H), 7.33 (t, J=8.08 Hz, 1H),6.98-7.02 (m, 1H), 6.89 (ddd, J=7.83, 2.27, 1.01 Hz, 1H), 3.92-4.01 (m,2H), 3.69 (br. s., 4H), 3.51 (s, 3H), 3.29-3.38 (m, 2H), 3.12 (s, 3H),2.99 (s, 3H), 2.43 (d, J=1.01 Hz, 3H), 2.31 (dt, J=13.64, 4.29 Hz, 2H),1.97-2.07 (m, 2H). MS (ES+) [M+H]⁺=497.3.

6.27. Example 27 Synthesis of3-(4-amino-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

A. Preparation of tert-butyl4-(tert-butoxycarbonylamino)-4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)piperidine-1-carboxylate.N-Boc-amino-(4-N-Boc-piperidinyl)carboxylic acid (0.114 g, 0.33 mmol),HATU (0.151 g, 0.40 mmol) and N,N-diisopropylethylamine (128 mg, 1.0mmol) were combined in isopropyl acetate (2.3 mL) and stirred at roomtemperature for 15 minutes. 3-aminophenyl dimethylcarbamate (0.072 g,0.40 mmol) was added, and the mixture heated at 80° C. for 1.5 hours.The reaction mixture was allowed to cool, diluted with EtOAc, washedwith 2 N Na₂CO₃, 1 N HCl, and brine, and then dried over MgSO₄ andconcentrated. Purification by silica gel chromatography (60%EtOAc/hexanes) gave the title compound (0.075 g, 45%) as a white solid.MS (ES+) [M+NH₄]⁺=524.

B. Preparation of 3-(4-aminopiperidine-4-carboxamido)phenyldimethylcarbamate. To a solution of tert-butyl4-(tert-butoxycarbonylamino)-4-(3-(dimethylcarbamoyloxy)phenylcarbamoyl)piperidine-1-carboxylatefrom step A (0.141 g, 0.28 mmol) in MeOH (2 mL) was added HCl (4 N indioxane, 0.21 mL, 0.84 mmol). The mixture was stirred at roomtemperature for 14 hours. Further eqivalents of HCl/dioxane were addedat this time, and the mixture stirred for a further 24 hours at roomtemperature. The solvent was removed under vacuum to afford the titlecompound (0.084 g, 98%), which was carried on crude. MS (ES+)[M+H]⁺=307.

C. Preparation of3-(4-amino-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate. 3-(4-aminopiperidine-4-carboxamido)phenyldimethylcarbamate from step B (0.109 g, 0.36 mmol),4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (0.060 g, 0.36 mmol), andN,N-diisopropylethylamine (0.277 g, 2.14 mmol) were combined inisopropanol (5 mL) and heated in a sealed tube at 120° C. for 18 hours.The mixture was concentrated, and the residue was purification by silicagel chromatography (0-10% MeOH/CH₂Cl₂) to afford the title compound(0.085 g, 55%) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.25 (s, 1H), 7.61 (m, 1H), 7.47 (m, 1H),7.38 (m, 1H), 7.05 (d, J=1.0 Hz, 1H), 6.91 (m, 1H), 4.00 (m, 2H), 2.48(m, 2H), 3.18 (s, 3H), 3.05 (s, 3H), 2.51 (s, 3H), 2.45 (m, 2H), 1.72(m, 2H); MS (ES+) [M+H]⁺=438.

6.28. Example 28 Synthesis of3-(4-(dimethylamino)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

3-(4-amino-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate from example 27, step C, (0.028 g, 0.064 mmol) wasdissolved in MeOH (1 mL). Formaldehyde (37 wt % solution in H₂O, 0.15mmol, 12 μl) was added followed by AcOH (0.26 mmol, 15 μl) and NaBH₃CN(0.008 g, 0.13 mmol). The mixture was stirred at room temperature for 2hours before concentrating under vacuum. Purification by prep HPLCafforded the title compound (0.017 g, 57%) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ ppm 8.21 (s, 1H), 7.56 (m, 1H), 7.46 (m, 1H),7.37 (m, 1H), 7.03 (d, J=1.0 Hz, 1H), 6.92 (m, 1H), 4.08 (m, 2H), 3.47(m, 2H), 3.17 (s, 3H), 3.04 (s, 3H), 2.48 (s, 3H), 2.40 (s, 6H), 2.26(m, 2H), 2.09 (m, 2H); MS (ES+) [M+H]⁺=466.

6.29. Example 29 Synthesis of3-(4-((2-(1H-imidazol-5-yl)acetamido)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate

To a suspension of 2-(1H-imidazol-5-yl)acetic acid (0.238 g, 1.47 mmol)and HATU (0.372 g, 0.978 mmol) in CH₂Cl₂ (5 mL) was addedN,N-diisopropylethylamine (0.51 mL, 2.93 mmol). The resulting solutionwas allowed to stir for 20 min. at room temperature. To this solutionwas added the acetate salt of3-(4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyldimethylcarbamate (from example 20, 0.500 g, 0.978 mmol) andN,N-diisopropylethylamine (0.17 mL) in CH₂Cl₂ (5 mL). The reaction wasallowed to stir at room temperature for 6h, and then diluted with H₂O(10 mL) and CH₂Cl₂ (20mL). The mixture was extracted with CH₂Cl₂ (2×10mL), and the combined organics were washed with NaHCO₃ (10 mL) and brine(10 mL), dried over MgSO₄, and concentrated under vacuum. The residuewas purified by flash chromatography (3-10% MeOH:CH₂Cl₂) to afford thetitle compound (0.393 g, 72% yield) as a white solid. This solid wassuspended in water, and acetic acid was added until the solution becameclear. This solution was frozen and lyophilized to the di-acetate saltof the title compound (0.477 g).

¹H NMR (400 MHz, MeOD-d₆) δ ppm 8.17 (s, 1H), 7.57 (d, J=0.76 Hz, 1H),7.47 (t, J=2.02 Hz, 1H), 7.37 (ddd, J=1.01, 1.77, 8.08 Hz, 1H), 7.30 (t,J=8.08 Hz, 1H), 6.98 (d, J=1.01 Hz, 1H), 6.92 (s, 1H), 6.87 (ddd,J=1.01, 2.27, 8.08 Hz, 1H), 3.87 (ddd, J=3.28, 3.28, 14.15 Hz, 2H), 3.58(s, 2H), 3.52 (s, 2H), 3.32-3.38 (m, 2H), 3.12 (s, 3H), 3.00 (s, 3H),2.42 (d, J=0.76 Hz, 3H), 2.35-2.42 (m, 2H), 1.96 (s, 6H), 1.75-1.85 (m,2H). MS (ES+) [M+H]=560.

6.30. Example 30 Additional Compounds

Using the procedures described herein and methods known in the art, theadditional compounds listed below in Table 1 were prepared. The LIMK2IC₅₀ data that is provided below for some of the compounds wasdetermined using the assay described in Example 32.

TABLE 1 MS Compound (M + H)⁺ LIMK2 IC₅₀N-(3-(4-fluorophenoxy)phenyl)-4-methyl-1-(5-methyl-7H- 460 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamideN-(3-bromophenyl)-4-methyl-1-(5-methyl-7H-pyrrolo[2,3- 428 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamideN-(3-bromophenyl)-1-(5-chloro-7H-pyrrolo[2,3- 450 <250 nMd]pyrimidin-4-yl)-4-methylpiperidine-4-carboxamideN-(3-(N-isopropylsulfamoyl)phenyl)-4-methyl-1-(5- 471 <250 nMmethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide4-allyl-N-(3-bromophenyl)-1-(5-methyl-7H-pyrrolo[2,3- 456 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamideN-(4-methoxyphenyl)-4-methyl-1-(5-methyl-7H- 380 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide4-allyl-N-(3-tert-butylphenyl)-1-(5-methyl-7H-pyrrolo[2,3- 432 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamide4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)- 418 <250 nMN-(3-(trifluoromethyl)phenyl)piperidine-4-carboxamide4-allyl-N-(3-(4-fluorophenoxy)phenyl)-1-(5-methyl-7H- 486 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide4-allyl-N-(3-fluorophenyl)-1-(5-methyl-7H-pyrrolo[2,3- 394 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamide4-allyl-N-(3-methoxyphenyl)-1-(5-methyl-7H-pyrrolo[2,3- 406 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamide3-(4-allyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4- 463 <250 nMyl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-isopropyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4- 465 <250 nMyl)piperidine-4-carboxamido)phenyl dimethylcarbamate4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)- 350 <250 nMN-phenylpiperidine-4-carboxamide3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4- 465 <250 nMpropylpiperidine-4-carboxamido)phenyl dimethylcarbamateN-(3-bromo-4-fluorophenyl)-4-methyl-1-(5-methyl-7H- 447 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)- 499 <250 nMN-(3-(morpholinosulfonyl)phenyl)piperidine-4- carboxamide4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)- 364 <250 nMN-m-tolylpiperidine-4-carboxamideN-(3-isobutyramidophenyl)-4-methyl-1-(5-methyl-7H- 435 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamideN-(3-(2-(dimethylamino)-2-oxoethyl)phenyl)-4-methyl-1- 435 <250 nM(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamide3-(4-methyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4- 479 <250 nMyl)piperidine-4-carboxamido)phenyl morpholine-4- carboxylate3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-((3- 509methylureido)methyl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-((2-(dimethylamino)ethoxy)methyl)-1-(5-methyl-7H- 524pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(4-cyano-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-448 yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-(acetamidomethyl)-1-(5-methyl-7H-pyrrolo[2,3- 494 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-(2-aminoethylamino)-1-(5-methyl-7H-pyrrolo[2,3- 481 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-(2-(tert-butoxycarbonylamino)ethylamino)-1-(5- 581 <250 nMmethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(4-(2-(benzyloxy)ethylamino)-1-(5-methyl-7H- 572<250 nM pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(4-cyano-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-448 <250 nM yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-(2-hydroxyethylamino)-1-(5-methyl-7H-pyrrolo[2,3- 482 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-((1-benzyl-1H-tetrazol-5-yl)methyl)-1-(5-methyl-7H- 595 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(4-((1H-tetrazol-5-yl)methyl)-1-(5-methyl-7H- 505<250 nM pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4- 530<250 nM (methylsulfonamidomethyl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(4-((N,N-dimethylsulfamoylamino)methyl)-1-(5-methyl-559 <250 nM 7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-((methoxycarbonylaminomethyl)-1-(5-methyl-7H- 510 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate3-(4-((1-methyl-1H-pyrazole-4-carboxamido)methyl)-1-(5- 560 <250 nMmethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4- 565<250 nM ((morpholine-4-carboxamido)methyl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4- 495 <250 nM(ureidomethyl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-((3-hydroxy-2-(hydroxymethyl)-2- 568 <250 nMmethylpropanamido)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-((1-tert- 635 <250 nMbutoxycarbonylaminocyclopropanecarboxamido)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-((2-aminoacetamido)methyl)-1-(5-methyl-7H- 509 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(4-((2-(dimethylamino)acetamido)methyl)-1-(5-methyl-537 <250 nM 7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4- 531 <250 nM((sulfamoylamino)methyl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-(2H-491 <250 nM tetrazol-5-yl)piperidine-4-carboxamido)phenyldimethylcarbamate 3-(4-((1H-imidazol-2-yl)methyl)-1-(5-methyl-7H- 503<250 nM pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(4-((1-aminocyclopropanecarboxamido)methyl)-1-(5-535 <250 nM methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate(S)-3-(4-((2-amino-3-(1H-imidazol-4- 589 <250 nMyl)propanamido)methyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin- 432 <250 nM4-yl)-N-(3-(oxazol-5-yl)phenyl)piperidine-4-carboxamide3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4- 522 <250 nM(morpholinomethyl)piperidine-4-carboxamido)phenyl dimethylcarbamate(S)-3-(4-((2-amino-3-hydroxypropanamido)methyl)-1-(5- 539 <250 nMmethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate (S)-3-(4-((2,5-diaminopentanamido)methyl)-1-(5-methyl-566 <250 nM 7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-(azidomethyl)-1-(5-methyl-7H-pyrrolo[2,3- 478 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate4-(aminomethyl)-N-(3-hydroxyphenyl)-1-(5-methyl-7H- 381 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-(2- 536 <250 nMmorpholinoethyl)piperidine-4-carboxamido)phenyl dimethylcarbamatedimethyl 1-((4-(3- 620 <250 nM(dimethylcarbamoyloxy)phenylcarbamoyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)methyl)-1H-1,2,3-triazole-4,5-dicarboxylate3-(4-((5-methyl-1H-pyrazole-3-carboxamido)methyl)-1-(5- 560 <250 nMmethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(4-((2-(2-methoxyethoxy)acetamido)methyl)-1-(5- 568<250 nM methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin- 379 <250 nM4-yl)-N-m-tolylpiperidine-4-carboxamide4-(aminomethyl)-N-(3-chlorophenyl)-1-(5-methyl-7H- 399 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide4-(aminomethyl)-N-(3-methoxyphenyl)-1-(5-methyl-7H- 395 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide4-(aminomethyl)-N-(3-fluorophenyl)-1-(5-methyl-7H- 383 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide4-(aminomethyl)-N-(3-(isopropylcarbamoyl)phenyl)-1-(5- 450 <250 nMmethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamide4-(aminomethyl)-N-(3-(dimethylcarbamoyl)phenyl)-1-(5- 436 <250 nMmethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamide4-(aminomethyl)-N-(3-(2-(dimethylamino)-2- 466 <250 nMoxoethoxy)phenyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide3-(4-((3-methoxypropanamido)methyl)-1-(5-methyl-7H- 538 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(4-((2-methoxyacetamido)methyl)-1-(5-methyl-7H- 524<250 nM pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldimethylcarbamate 4-(aminomethyl)-N-(3-(2-methoxyethoxy)phenyl)-1-(5-439 <250 nM methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide 4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin- 433<250 nM 4-yl)-N-(3-(trifluoromethyl)phenyl)piperidine-4- carboxamide4-(aminomethyl)-N-(3-cyanophenyl)-1-(5-methyl-7H- 390 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide3-(4-((2-(2,5-dioxoimidazolidin-4-yl)acetamido)methyl)-1- 592 <250 nM(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl dimethylcarbamate4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin- 435 <500 nM4-yl)-N-(2-(methylamino)benzo[d]oxazol-6-yl)piperidine- 4-carboxamide4-(aminomethyl)-N-(2-(dimethylamino)benzo[d]oxazol-6- 449 <500 nMyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide3-(4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3- 480 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl diethylcarbamate3-(4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3- 478 <250 nMd]pyrimidin-4-yl)piperidine-4-carboxamido)phenylpyrrolidine-1-carboxylate4-(aminomethyl)-N-(3-bromophenyl)-1-(5-methyl-7H- 443 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide4-(aminomethyl)-N-(3-isopropoxyphenyl)-1-(5-methyl- 423 <250 nM7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamide4-(aminomethyl)-N-(3-tert-butylphenyl)-1-(5-methyl-7H- 421 <250 nMpyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide(R)-3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4- 549((pyrrolidine-2-carboxamido)methyl)piperidine-4- carboxamido)phenyldimethylcarbamate 3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4- 466((methylamino)methyl)piperidine-4-carboxamido)phenyl dimethylcarbamate3-(4-(2-(dimethylamino)ethyl)-1-(5-methyl-7H- 522pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamido)phenyldiethylcarbamate 4-(2-(dimethylamino)ethyl)-N-(3-isopropoxyphenyl)-1-(5-465 methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamideN-(3-tert-butylphenyl)-4-(2-(dimethylamino)ethyl)-1-(5- 463methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamide3-(1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-((5- 563oxopyrrolidine-2-carboxamido)methyl)piperidine-4- carboxamido)phenyldimethylcarbamate N-(3-bromophenyl)-4-(2-(dimethylamino)ethyl)-1-(5- 485methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamide4-(2-(dimethylamino)ethyl)-1-(5-methyl-7H-pyrrolo[2,3- 475d]pyrimidin-4-yl)-N-(3-(trifluoromethyl)phenyl)piperidine- 4-carboxamide3-(4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3- 508d]pyrimidin-4-yl)piperidine-4-carboxamido)phenyl diisopropylcarbamate4-(aminomethyl)-N-(4-(aminomethyl)phenyl)-1-(5-methyl- 3947H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamide4-(aminomethyl)-N-(4-((dimethylamino)methyl)phenyl)-1- 422(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamide4-(aminomethyl)-N-(3-ethoxyphenyl)-1-(5-methyl-7H- 409pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide4-(aminomethyl)-N-(3-isobutoxyphenyl)-1-(5-methyl-7H- 437pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide(R)-4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3- 451d]pyrimidin-4-yl)-N-(3-(tetrahydrofuran-3-yloxy)phenyl)piperidine-4-carboxamide4-(aminomethyl)-N-(3-(cyclopentyloxy)phenyl)-1-(5- 449methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamide4-(aminomethyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin- 4654-yl)-N-(3-(tetrahydro-2H-pyran-4- yloxy)phenyl)piperidine-4-carboxamide4-(aminomethyl)-N-(3-((3R,4R)-4- 467hydroxytetrahydrofuran-3-yloxy)phenyl)-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide

6.31. Expression and Purification of LIMK2

LIMK2 was expressed using the BAC-to-BAC® Baculovirus Expression System(Invitrogen). Recombinant baculovirus was made according to themanufacturer's directions as set forth in the instruction manual.Briefly, the plasmids (pFactBac1 or pFastBacHT) carrying the LIMK2inserts were transformed into MAX efficiency DH10Bac competent E. colito generate a recombinant bacmid. The DH10Bac E. coli host straincontains a baculovirus shuttle vector (bacmid) with a mini-attTn7 targetsite and a helper plasmid, and allows generation of a recombinant bacmidfollowing transposition between the mini-Tn7 element on the pFastBacvector and the min-attTn7 target site on the bacmid. The transpositionreaction occurs in the presence of transposition proteins supplied bythe helper plasmid. Cells were plated and the white colonies picked forbacmid isolation as described in the instruction manual.

The isolated bacmid DNA was transfected into SF9 cells to generate arecombinant baculovirus, and virus was collected five days aftertransfection. Virus was amplified in T75 flasks at a multiplicity ofinfection (MOI) of 0.2. The amplified virus was used to infect SF9 cellsat a MOI 5 for protein expression.

For small scale purification of the LIMK2 constructs, a 50 ml culture ofSf9 cells infected with the recombinant baculovirus was used. The cellswere harvested by centrifugation for 5 minutes at 500×g. The cells werethen resuspended in lysis buffer (5 volumes per gram of cells). Atypical lysis buffer contains the following: 50 mM HEPES (pH 8.0), 300mM KCl, 10% glycerol, 1% NP-40, 15mM imidazole, 1 mM benzamidine, andRoche complete protease inhibitors (1 tablet per 50 ml of cell lysate).The cellular suspension was lysed by one passage through a MicrofluidicsMicrofluidizer M-110Y at a liquid pressure of 14,000 to 20,000 psifollowed by centrifugation of the lysate at 60,000×g for 15 minutes at4° C.

The supernatant was then loaded directly onto a chromatography matrixcontaining Cobalt ion covalently attached to nitrilotriacetic acid NTA.The chromatography matrix was equilibrated in the same buffer as theprotein loading solution. The ion charged resin typically has a bindingcapacity equivalent to 5 to 10 mg histidine-tagged protein per ml ofpacked resin. The amount of extract that can be loaded onto the columndepends on the amount of soluble histidine-tagged protein in theextract. The column was then washed in a stepwise fashion, first with:50 mM HEPES (pH 8.0), 300 mM KCl, 10% glycerol, 1% NP-40, 15mMimidazole, 1 mM benzamidine; second, with 20 mM HEPES (pH 8.0), 500 mMKCl, 10% glycerol, and 20 mM imidazole; third, with 20 mM HEPES (pH8.0), 100 mM KCl, 10% glycerol, and 20 mM imidazole; followed by elutionwith 250 mM imidazole in the same buffer. The LIMK2 protein solution wasthen analyzed by SDS-PAGE and Western blot using commercial antibodiesdirected to both the carboxyl terminus and internal catalytic domains ofthe protein. For storage purposes the protein was dialyzed into 50 mMTris (pH 7.5), 150 mM NaCl, 0.1% BME, 0.03% Brij-35, and 50% glycerol.

Large scale LIMK2 purification was done in a Wave Bioreactor (WaveBiotech) with 10 L culture volumes. 10 L of cell culture at 2-3×10⁶viable cells/mL were infected at an MOI=5 pfu/cell and harvested at 48hours post infection.

6.32. In Vitro LIMK2 Inhibition Assay

An in vitro assay used to identify LIMK2 inhibitors was developed. Theanalytical readout was the incorporation of ³³P from ATP substrate intoimmobilized myelin basic protein coated flash plates (Perkin ElmerBiosciences), which were counted on a scintillation counter equippedwith a plate reader (TopCount, Packard Bioscience, Meriden, Conn.).Using 384 well flat MBP flashplates, total assay volume was 50 μl. TheHTS program utilized a Biomek FX for dilution.

For each assay, the ingredients and conditions were as follows: 200 ngof enzyme was incubated in assay buffer (1× assay buffer contains 30 mMHEPES (pH 8.0), 5 mM DTT, and 10 mM MgCl₂), 10 μM ATP, 0.2 μCi[gamma-³³P]-ATP and 10 μM of potential inhibitory compound. The reactionwas incubated at room temperature for 60 minutes, washed 3 times with 75μl of stop/wash buffer (1× stop/was buffer contains 50 mM EDTA and 20 mMTris (pH 7.4)), and then the plates were read on the scintillationcounter. Different concentrations of staurosporine (400 nM, 200 nM, 100nM and 50 nM; purchased from BIOMOL (Plymouth Meeting, Pa.)) were usedas controls on each plate.

6.33. Dexamethasone-Induced Ocular Hypertension Model

Twenty eight day mouse Alzet mini-osmotic pumps (DURECT Corp.,Cupertino, Calif.) were filled with a solution of water solubledexamethasone (dex) in PBS (Sigma, St. Louis, Mo.) so that they wouldrelease roughly 0.1 mg of dex per day. Once the pumps were filled withthe dex, the pumps were allowed to equilibrate in PBS at 37° C. for 60hours. The equilibrated pumps were surgically placed subcutaneously onthe backs of wild-type C57:129 F2 hybrid mice weighing between 25 and 35grams. Surgical incisions were sutured with 5-0 braided silk (ROBOZ,Gaithersburg, Md.) and treated with antibiotic ointment throughout theentire duration of study. Surgical incisions were glued with TissueMendII (Webster Veterinary, Houston, Tex.). Analgesic (buprenorphine) wasgiven through IP injection the day of surgery and 24 hours aftersurgery. Intraocular pressure (IOP) was measured on these mice using aTonoLab (Colonial Medical Supply Co., Franconia, N.H.) tonometer. Micewere mildly sedated with isoflurane and topically anesthetized with 0.5%proparacaine (Akorn, Buffalo Grove, Ill.) before IOP measurements weretaken. Baseline IOP was measured 1 day prior to mini-pump implantation.After mini-pump implantation, IOP measurements were taken 2-3 times perweek for 4 weeks. Pharmacology studies with potential ocular hypotensivecompounds were performed between 21 and 28 days after implantation.

6.34. In Vivo Effects

Compounds of the invention were then tested in the mouse ocularhypertensive model, described above. The effects of four compounds ofthe invention topically administered to the eyes of the mice aredescribed below in Table 2:

TABLE 2 Change in IOP (Number of Studies)^(c) Compound Dose^(a)Formulation^(b) 2 hours 4 hours 6 hours A 30 1 −4.4 −1.6 Not tested A 31 −4.2 (7) −2.6 (7) −1.3 (4) A 0.3 1 −2.0 (3) −1.6 (3) −0.1 A 3 2 −4.6−2.6 −1.4 B 30 2 −4.1 −2.5 −.03 B 3 2 −3.4 −2.2 −0.3 C 3 3 −3.5 (2) −0.9(2) −0.5 (2) D 3 3 −1.8 −0.8 Not tested D 0.3 3 −2.2 −0.4 Not tested^(a)Microgram per eye. ^(b)Ophthalmically acceptable excipients wereused to make different formulations. The pH of formulation 1 was 7.2;the pH of formulation 2 was 7.2; and the pH of formulation 3 was 6.0.^(c)Times are hours after dosing.

The effect of compound A was compared to that of timolol in thehypertensive model. As shown in FIG. 1, the topical administration of 3μg per eye of compound A yielded substantially the effect as the topicaladministration of 15 μg per eye of timolol.

All publications (e.g., patents and patent applications) cited above areincorporated herein by reference in their entireties.

1. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein: A is optionallysubstituted aryl or heterocycle; R₁ is hydrogen, halogen, cyano, oroptionally substituted alkyl, heteroalkyl, aryl, or heterocycle; R₂ ishydrogen, halogen, cyano, or optionally substituted alkyl, heteroalkyl,aryl, or heterocycle; R₃ is R_(3A), OR_(3B), N(R_(3B))₂, NC(O)R_(3B),NC(O)OR_(3B), NC(O)N(R_(3B))₂, SR_(3B), SOR_(3B), or SO₂R_(3B); eachR_(3A) is cyano, C(O)R_(3C), CO₂R_(3C), CON(R_(3C))₂, or optionallysubstituted alkyl, alkylaryl, alkylheterocycle, aryl, heteroalkyl,heteroalkylaryl, heteroalkylheterocycle, or heterocycle; each R_(3B) isindependently hydrogen, C(O)R_(3C), CO₂R_(3C), CON(R_(3C))₂, oroptionally substituted alkyl, heteroalkyl, aryl, or heterocycle; eachR_(3C) is independently optionally substituted alkyl, aryl orheterocycle; each R₄ is independently halogen, OR_(4A), OC(O)R_(4A),OC(O)N(R_(4A))₂, N(R_(4A))₂, NC(O)R_(4A), NC(O)OR_(4A), NC(O)N(R_(4A))₂,SR_(4A), SOR_(4A), SO₂R_(4A), or optionally substituted alkyl,heteroalkyl, aryl, or heterocycle; each R_(4A) is independently hydrogenor optionally substituted alkyl or heteroalkyl; and n is 0-3; with theproviso that when R₁ and R₂ are both hydrogen and A is phenyl, R₄ is nothalogen. 2-26. (canceled)
 27. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein: R₁ is hydrogen,halogen, cyano, or optionally substituted alkyl, heteroalkyl, aryl, orheterocycle; R₃ is R_(3A), OR_(3B), N(R_(3B))₂, NC(O)R_(3B),NC(O)OR_(3B), NC(O)N(R_(3B))₂, SR_(3B), SOR_(3B), or SO₂R_(3B); eachR_(3A) is cyano, C(O)R_(3C), CO₂R_(3C), CON(R_(3C))₂, or optionallysubstituted alkyl, alkylaryl, alkylheterocycle, aryl, heteroalkyl,heteroalkylaryl, heteroalkylheterocycle, or heterocycle; each R_(3B) isindependently hydrogen, C(O)R_(3C), CO₂R_(3C), CON(R_(3C))₂, oroptionally substituted alkyl, heteroalkyl, aryl, or heterocycle; eachR_(3C) is independently optionally substituted alkyl, aryl orheterocycle; R₄ is halogen, OR_(4A), OC(O)R_(4A), OC(O)N(R_(4A))₂,N(R_(4A))₂, NC(O)R_(4A), NC(O)OR_(4A), NC(O)N(R_(4A))₂, SR_(4A),SOR_(4A), SO₂R_(4A), or optionally substituted alkyl, heteroalkyl, aryl,or heterocycle; and each R_(4A) is independently hydrogen or optionallysubstituted alkyl or heteroalkyl.
 28. (canceled)
 29. (canceled)
 30. Thecompound of claim 27, wherein R₁ is hydrogen, halogen, cyano, oroptionally substituted lower alkyl.
 31. The compound of claim 30,wherein R₁ is hydrogen.
 32. The compound of claim 30, wherein R₁ ishalogen.
 33. The compound of claim 30, wherein R₁ is optionallysubstituted lower alkyl.
 34. The compound of claim 30, wherein R₃ isR_(3A), OR_(3B), or N(R_(3B))₂.
 35. The compound of claim 34, wherein R₃is R_(3A) and R_(3A) is optionally substituted alkyl, alkylheterocycle,heteroalkyl, or heterocycle.
 36. The compound of claim 35, whereinR_(3A) is optionally substituted alkyl.
 37. The compound of claim 35,wherein R_(3A) is optionally substituted alkylheterocycle.
 38. Thecompound of claim 35, wherein R_(3A) is optionally substitutedheteroalkyl.
 39. The compound of claim 35, wherein R_(3A) is optionallysubstituted heterocycle.
 40. The compound of claim 34, wherein R₃ isOR_(3B) or N(R_(3B))₂, and R_(3B) is hydrogen or optionally substitutedalkyl or aryl.
 41. The compound of claim 40, wherein R_(3B) is hydrogen.42. The compound of claim 40, wherein R_(3B) is optionally substitutedalkyl.
 43. The compound of claim 40, wherein R_(3B) is optionallysubstituted aryl.
 44. The compound of claim 27, wherein R₄ is halogen.45. The compound of claim 27, wherein R₄ is OC(O)N(R_(4A))₂.
 46. Thecompound of claim 45, wherein at least one R_(4A) is lower alkyl.
 47. Apharmaceutical formulation comprising a liquid vehicle suitable forophthalmic administration and a compound of claim
 1. 48. A method oflowering intraocular pressure in a patient, which comprisesadministering to a patient in need thereof a therapeutically orprophylactically effective amount of a compound of claim
 1. 49. A methodof treating, managing or preventing a disease or disorder affectingvision in a patient, which comprises administering to a patient in needthereof a therapeutically or prophylactically effective amount of acompound of claim
 1. 50. (canceled)
 51. (canceled)
 52. (canceled)